Pyrazoloanthrone and derivatives thereof for the treatment of cancer expressing &#39;mullerian inhibiting substance&#39; type ii receptor (misrii) and of excess androgen states

ABSTRACT

The present invention relates to pyrazoloanthrones or functional derivatives or functional analogues thereof to activate MIS receptor-mediated downstream effects in a cell. In particular, the present invention relates to method to prevent and treat cancer that expresses MIS receptor type II (MISRII) by administering to a subject at least one pyrazoloanthrone or a functional derivative or a functional analogue thereof. Another aspect of the present invention relates to methods to lower plasma androgen levels in a subject, and/or for the treatment of a subject with a disease characterized by excess androgen, whereby the subject is administered at least one pyrazoloanthrone or a functional derivative or a functional analogue thereof. Another aspect provides pharmaceutical compositions comprising at least one pyrazoloanthrone or functional a derivative or a functional analogue thereof, and optionally with one or more additional agents such as chemotherapeutic agents. Another aspect of the present invention relates to methods to decrease the dose of a chemotherapeutic agent by administering the chemotherapeutic agent with a pyrazoloanthrone or a functional derivative or a functional analogue thereof that lowers the effective dose of the chemotherapeutic agent, such as for example, paclitaxel.

CROSS REFERENCED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) of U.S.Provisional Application Ser. No. 60/919,429, filed on Mar., 22, 2007 thecontents of which are incorporated herein in their entirety byreference.

GOVERNMENT SUPPORT

This application was made with government support under Grant NoCA017393-30S1 awarded by the National Institutes for Health (NIH). TheGovernment of the United States has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer is characterized by uncontrolled growth, proliferation, andmigration of cells. Cancer is the second leading cause of death with500,000 deaths and an estimated 1.3 million new cases in the UnitedStates in 1996. The role of signal transduction pathways contributing tocell transformation and cancer is a generally accepted concept.

Müllerian Inhibiting Substance (MIS) is a glycoprotein hormone secretedby the newly differentiating testis during the fetal period where it isresponsible for regression of the Müllerian ducts in males, which wouldotherwise develop into the internal female reproductive tract tissues(Reviewed in (Teixeira, 2001)). However, MIS is also produced duringpostnatal life in both male and female gonads (Takahashi, 1986, Tran,1987). As a member of the transforming growth factor-β (TGF-β) family,MIS signaling requires a set of membrane bound serine/threonine kinasereceptors (Baarends, 1994, Clarke, 2001a, di Clemente, 1994, He, 1993,Jamin, 2002, Teixeira, 1996, Visser, 2001). After the type II receptorfor MIS (MISRII) binds the MIS ligand, it recruits, phosphorylates, andactivates one of three possible, activin-like kinase type I receptors(Alk2 or 3), which in turn activates the Smad 1/5/8 pathway in concertwith the common Smad4. The Smad complex will translocate into thenucleus and bind to promoter regions to activate transcription ofMIS-responsive genes (Massague and Wotton, 2000).

In addition to its role during male fetal development, the continuousproduction of this hormone in both male and females after birthindicates a function in the adult. One of the activities that has beenascribed to postnatal MIS has been as a inhibitor of tumor cellproliferation, including cells from breast (Gupta, 2005, Segev, 2000),prostate (Hoshiya, 2003, Tran, 2006), cervical (Barbie, 2003),endometrial (Renaud, 2005) and ovarian (Ha, 2000, Stephen, 2002)cancers. Since, the type II receptor for MIS is highly expressed only inthe gonads and other reproductive tract organs, targeting this receptorshould give rise to a selective agent and minimize side effectsassociated with other systemic therapy. MISRII is expressed in motorneurons (Wang, 2005) but the effect is salutary rather than inhibitoryas in tumors.

However, the production of large quantities of purified, biologicallyactive MIS sufficient to be used therapeutically is challenging.Therefore, it would be desirable to have a simple molecule that couldmimic the effect of MIS by selectively activating the MISRII-mediateddownstream signaling pathway.

SUMMARY OF THE INVENTION

The inventors have discovered that pyrazoloathrones or derivativesthereof act as a MIS mimetic. The present invention relates to the useof pyrazoloathrones or derivatives thereof to activate MISreceptor-mediated downstream effects that result in the inhibition oftumor cell proliferation, for example inhibition of MISRII positivetumors.

The present invention is based on the discovery that pyrazoloathrone orfunctional derivative thereof can mimic the effect of MIS by selectivelyactivating the MIS type II receptor (MISRII)-mediated downstreamsignaling pathway. The inventors have also discovered that in thepresence of pyrazoloathrone or derivative thereof, a lower effectiveconcentration of MIS and/or other agents can be used in the treatment ofcancers expressing MIS receptors, for example cancer expressing MISRII.Examples of such agents are, for example but not limited to,chemotherapy agents such as paclitaxel for the treatment of cancersexpressing MIS receptors, for example cancer expressing MISRII. In otherembodiments, the agent is a MIS mimetic. In some embodiments the cancerexpressing MIS receptors, for example cancer expressing MISRII is, forexample, ovarian cancer and cervical cancer.

Accordingly, one aspect of the present invention relates to methods forthe treatment of cancers expressing MIS receptors, for example cancerexpressing MISRII. In some embodiments, the subject is at risk ofdeveloping, or has a cancer expressing MIS receptors, for example thesubject is at risk of developing or has a cancer expressing MISRII. Thepresent invention provides methods to treat cancer expressing MISreceptors, for example cancer expressing MISRII by administering to thesubject a pharmaceutical composition comprising a pyrazoloathrone orderivative or analogue thereof. The present invention relates to amethod to treat cancers expressing MIS receptors, for example cancerexpressing MISRII, the method comprising contacting a cell with apyrazoloathrone or derivative or analogue thereof. In some embodimentsthe pyrazoloathrone is antra(1,9-cd)pyrazol-6(2H)-one or an analogue orderivative thereof. In some embodiments, the cell, for example a cancercell expressing MISRII is also contacted with agents in addition to thepyrazoloathrone or derivative or analogue thereof, and in someembodiments, the agents are therapeutic agents and/or chemotherapeuticagents. The cell expressing MIS receptors or MISRII can be any cell ofany type. In some embodiments, the cell is a cancer cell expressing MISreceptors, for example a cancer cell expressing MISRII.

In another embodiment, a biological sample is obtained from the subjectand assessed for the expression of a MIS receptor, for example for theexpression of MISRII, and if the presence of expression and/or activityof the MIS receptor, for example MISRII is detected, the subject isadministered a pharmaceutical composition comprising a pyrazoloathroneor derivative or analogue thereof. In another embodiment, thepharmaceutical composition comprising pyrazoloathrone or derivative oranalogue thereof are administered with additional agents. Additionalagents include, for example but not limited to, therapeutic agentsand/or chemotherapeutic agents or MIS itself.

In another aspect of the present invention, the methods relate to theuse of pyrazoloathrone and functional derivatives thereof for thetreatment of any disorder where administration of the MIS protein or anucleic acid encoding MIS protein or activation of MISRII is whole, orpart, of the therapeutic regime. In some embodiments, thepyrazoloathrone is SP600125, as disclosed in U.S. Pat. No. 7,119,114which is specifically incorporated herein in its entirety by reference.In other embodiments, the pyrazoloathrone is a derivative ofpyrazoloathrone, for example substituted antra(1,9-cd)pyrazol-6(2H)-one,and anthrapyrazolone functional derivatives as disclosed in EuropeanPatent EP0103381 and EP0700390, which are incorporated herein in theirentirety by reference.

In some embodiments, the pyrazoloanthrone, for exampleantra(1,9-cd)pyrazol-6(2H)-one (herein also referred to as SP600125 orCompound I). In alternative embodiments, the pyrazoloanthrone is afunctional derivative of a pyrazoloanthrone, and may generally beclassified as “pyrazoloanthrone derivatives” having the followingstructure (I), also referred to herein as Compound (II):

wherein R₁ and R₂ are as defined below, including pharmaceuticallyacceptable salts thereof. Pyrazoloanthrone, for exampleantra(1,9-cd)pyrazol-6(2H)-one (herein also referred to as SP600125 orCompound II) and pyrazoloanthrone derivatives (having the structure (I)are disclosed in U.S. Pat. No. 7,119,114 which is incorporated in itsentirety herein by reference.

In some embodiments, the disorder is a proliferative disease where theproliferative disease is associated with cells expressing MIS receptors,for example cells expressing MISRII. One example of such a proliferativedisease is, for example a cancer expressing MIS receptors or a cancerexpressing MISRII. In some embodiments, the cancer is also aMIS-responsive cancer, for example but not limited ovarian cancer andcervical cancer. In some embodiments, the cancer expresses MISRII, forexample but not limited ovarian cancer and cervical cancer. In someembodiments, the disorder is a disorder associated with excess androgenstates, for example as disclosed in U.S. Pat. No. 6,673,352, which isincorporated in its entirety herein by reference. In some embodiments,the methods of the present invention are used in the treatment ofprostatic cancer, polycysic ovarian disease, benign prostatichypertrophy and precocious puberty.

The present invention is also directed towards methods for treatingcancers expressing MIS receptors, for example cancer expressing MISRIIby administering an effective amount of the pyrazoloathrone andfunctional derivatives thereof to a subject in need thereof. Accordinglythe compounds of the present invention are useful in treatment ofcancers expressing the MIS type II receptor (MISRII), for exampleovarian cancers. The compounds of the present invention are also usefulin treatment of other cancers expressing MIS receptors, for exampleother cancer expressing MISRII, for example cervical, breast, andprostate cancer. In some embodiments, the cancer is a cancer cellexpressing MIS receptors, for example cancer expressing MISRII. In someembodiments, the cancer cell expressing MIS receptors and/or MISRII isan ovarian cancer cell, vulvar epidermal carcinoma cell, cervicalcarcinoma cell, endometrial adenocarcinoma cell and ovarianadenocarcinoma.

In alternative embodiments, the cancer expressing MIS receptors, forexample cancer expressing MISRII is, for example but not limited tobreast cancer, lung cancer, head and neck cancer, bladder cancer,stomach cancer, cancer of the nervous system, bone cancer, bone marrowcancer, brain cancer, colon cancer, esophageal cancer, endometrialcancer, gastrointestinal cancer, genital-urinary cancer, stomach cancer,lymphomas, melanoma, glioma, bladder cancer, pancreatic cancer, gumcancer, kidney cancer, retinal cancer, liver cancer, nasopharynx cancer,ovarian cancer, oral cancers, bladder cancer, hematological neoplasms,follicular lymphoma, cervical cancer, multiple myeloma, osteosarcomas,thyroid cancer, prostate cancer, colon cancer, prostate cancer, skincancer, stomach cancer, testis cancer, tongue cancer, or uterine cancer.

Accordingly, one aspect of the present invention relates to a method fortreating a cancer in a subject, the method comprising administering tothe subject an effective amount of a pharmaceutical compositioncomprising a pyrazoloanthrone or a functional derivative or a functionalanalogue thereof, wherein the subject is determined to have a cancerexpressing a Mullerian Inhibiting Substance (MIS) receptor. In someembodiments, the pyrazoloanthrone is anthra[1,9-cd]pyrazol-6(2H)-one orfunctional derivative or functional analogue thereof, and the cancerexpresses the MIS Type II receptor (MISRII) or a homologue or functionalfragment thereof.

In some embodiments, a biological sample is harvested from the subjectto determine if the cancer expresses MIS receptor, and in someembodiments, the biological sample is a tissue sample, for example acancer or tumor tissue sample or a cancer cell or tumor cell, or abiopsy tissue sample.

In some embodiments, the methods to treat a cancer as disclosed hereinare useful for the treatment where the cancer comprises, for example butnot limited to, an ovarian cancer cell, a vulvar epidermal carcinomacell, a cervical carcinoma cell, an endometrial edenocarinaoma celland/or an ovarian adenocarcinoma cell. In alternative embodiments, themethods to treat a cancer as disclosed herein are useful for thetreatment of cancers such as, but not limited to, breast cancer, lungcancer, head and neck cancer, bladder cancer, stomach cancer, cancer ofthe nervous system, bone cancer, bone marrow cancer, brain cancer, coloncancer, esophageal cancer, endometrial cancer, gastrointestinal cancer,gum cancer, kidney cancer, liver cancer, nasopharynx cancer, ovariancancer, prostate cancer, skin cancer, stomach cancer, testis cancer,tongue cancer, or uterine cancer. In some embodiments, the cancer is amulti-drug resistant cancer, for example, a paclitaxel-resistant cancer.

In some embodiments in the methods to treat a cancer as disclosedherein, one can measure the expression of the nucleic acid gene productor protein or polypeptide gene product of the MIS receptor.

In some embodiments in the methods to treat a cancer as disclosedherein, one can administer the pyrazoloanthrone or a functionalderivative or a functional analogue thereof by intravenous, intradermal,intramuscular, intraarterial, intralesional, percutaneous, subcutaneous,or by aerosol. In some embodiments, the administering is prophylacticadministration, and alternative embodiments, the administering istherapeutic administration.

In some embodiments in the methods to treat a cancer as disclosedherein, the subject is a mammal, such as for example but not limited to,a human.

In some embodiments in the methods to treat a cancer as disclosedherein, one or more additional agents can be administered to the subjectin addition to the pyrazoloanthrone or a functional derivative or afunctional analogue thereof, for example, where the agents aretherapeutic agents such as chemotherapeutic agents. In some embodiments,the chemotherapeutic agents include, but are not limited to, paclitaxel,cisplatin, doxorubicin or rapamycin. In alternative embodiments, anadditional agent can be, for example, Mullerian Inhibiting Substance(MIS) or a functional derivative or variant thereof, such as recombinanthuman MIS (rhMIS). In some embodiments, the additional agent is aradiotherapeutic agent.

In some embodiments in the methods to treat a cancer as disclosedherein, one can administer the pyrazoloanthrone or a functionalderivative or a functional analogue thereof more than once. In someembodiments, a pyrazoloanthrone or functional derivative or functionalanalogue can be administered before, after or at the same time as theadditional agent, and in some embodiments, an additional therapeuticagent can be administered more than once via any route commonly know bypersons of ordinary skill in the art, such as intravenous, intradermal,intramuscular, intraarterial, intralesional, percutaneous, subcutaneous,or by aerosol administration.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising at least one pyrazoloathrone or at least onefunctional derivatives thereof, which can can be used alone or incombination with additional agents. In alternative embodiments, thepharmaceutical composition comprising pyrazoloathrone and functionalderivatives thereof can be used in combination with other therapeuticagents and additional therapies. In some embodiments, the additionaltherapies are, for example but not limited to chemotherapy,radiotherapy, thermotherapy, immunotherapy, hormone therapy, surgery andlaser therapy.

In some embodiments, the present invention relates to a pharmaceuticalcomposition comprising an activator of MISRII and a pharmaceuticalacceptable carrier, for example where the activator of MISRII is apyrazoloanthrone or a functional derivative or functional analoguethereof, such as for example, a anthra[1,9-cd]pyrazol-6(2H)-one or afunctional derivative or functional analogue thereof. In someembodiments, the pharmaceutical composition can further comprise one ormore additional agents, such as therapeutic agents for example,chemotherapeutic agents and/or a radiotherapeutic agent. In someembodiments, a chemotherapeutic agents which can be included in thepharmaceutical composition can be, for example but are not limited to,paclitaxel, cisplatin, doxorubicin or rapamycin or analogues orfunctional derivatives thereof. In some embodiments, an additional agentwhich can be added to the pharmaceutical compositions as disclosedherein can be, for example, Mullerian Inhibiting Substance (MIS) or afunctional derivative or functional variant thereof, such as recombinanthuman MIS (rhMIS). Other examples of therapeutic agents which can beadded or administered to the subject in addition to the pharmaceuticalcompositions as disclosed herein can be, but are not limited to, arepaclitaxel, cisplatin, and any other or combination of chemotherapyagents commonly known by person of ordinary skill in the art. In someembodiments, the therapeutic agent is MIS and/or recombinant or modifiedversion of MIS, for example rhMIS, or a functional derivatives of MIS,as disclosed in International Patent Application WO92/18153, which isincorporated herein in its entirety by reference. In some embodiments,the other therapeutic agent is in interferon, for example as disclosedin U.S. Patent Application 2004/0151693, which is incorporated herein inits entirety by reference.

Accordingly, the methods of the present invention are directed to use ofpyrazoloathrone and functional derivatives thereof with othertherapeutic agents, for example chemotherapy agents, wherein thechemotherapy agents, for example paclitaxel and/or MIS can be used at alower dose as compared to when they are used in the absence of thepyrazoloathrone or functional derivative thereof. Accordingly, where thechemotherapeutic, such as paclitaxel or MIS are used at a lower dose,this often results in decreased side effects associated with use of suchchemotherapeutics such as paclitaxel or MIS.

Another aspect of the present invention relates to methods of increasingthe sensitivity of a tumor cell to chemotherapeutic agent, the methodcomprising administering to the cell a therapeutically effective amountof a pyrazoloanthrone or functional derivative or functional analoguethereof. A similar apect of the present invention relates to a method ofdecreasing the dose of a chemotherapeutic agent for the treatment ofcancer, the method comprising administering to the subject atherapeutically effective amount of a pyrazoloanthrone or functionalderivative or functional analogue thereof and a chemotherapeutic agent,wherein the therapeutically effective dose of the chemotherapeutic agentin the presence of the pyrazoloanthrone or functional derivative orfunctional analogue is lower or at a decreased dose as compared to thetherapeutically effective dose of the chemotherapeutic agent when usedalone or in the absence of the pyrazoloanthrone or functional derivativeor functional analogue thereof. In some embodiments, the apyrazoloanthrone which is used to increase the sensitivity of a tumorcell, and/or decrease the dose of a chemotherapeutic agents isanthra[1,9-cd]pyrazol-6(2H)-one or derivative or analogue thereof. Insome embodiments, such a chemotherapeutic agent is, for example, but notlimited to, paclitaxel, cisplatin, doxorubicin, rapamycin or functionalderivatives thereof. In alternative embodiments, the chemotherapeuticagent is Mullerian Inhibiting Substance (MIS) or a functional derivativeor variant thereof, such as, for example but not limited to, recombinanthuman MIS (rhMIS). In some embodiments, the pyrazoloanthrone orfunctional derivative or functional analogue thereof can be administeredto the subject or to the tumor cell at the same time, or prior to, orfollowing the administration of a chemotherapeutic agent, as disclosedherein.

In some embodiments, the present invention provides methods formanufacture of a medicament for reducing cancer where the cancer is aMIS-responsive cancer, where the medicament comprises a pharmaceuticalcomposition of pyrazoloathrone and functional derivatives thereof, withor without additional therapeutic agents, as discussed above.

In another aspect, the present invention is directed to methods ofscreening for agents or molecules that function as a mimetic of MIS oractivate the MISRII or activate MISRII-mediated signaling. In someembodiments, the present invention provides methods an assay, whereinthe agent is contacted with a cell comprising a nucleic acid constructcomprising a BMP-responsive element operatively linked to a nucleic acidencoding a reporter gene. In some embodiments, the reporter gene encodesa protein with fluorescent activity and/or chromogenic activity, forexample but not limited to fluorescent proteins, for example greenfluorescent protein (GFP) or variants thereof or bioluminescentproteins, for example luciferase or variants thereof. In someembodiments, the cell also comprises a nucleic acid construct comprisingMISRII. In some embodiments, the agent is a small molecule, nucleicacid, nucleic acid analogue, aptamer, ribosome, peptide, protein,antibody or variants or fragments thereof. An agent that functions as aMIS mimetic or functional derivative of MIS or activates MISRII-mediatedsignaling will result in a change in the signal from the reporter gene,where the change is a result of contacting the cell with agent comparedto when the cell is not contacted with the agent.

In some embodiments, the present invention provides methods of identifyan agent that modulates MISRII-mediated signal transduction in a cell,the methods comprising; (a) providing a cell containing a nucleic acidconstruct comprising a nucleic acid sequence encoding a BMP-responsiveelement (BME) or fragment thereof, which is operatively linked to anucleic acid sequence encoding a reporter gene; and (b) contacting thecell or an extract of the cell with an agent; and (c) measuring thesignal from the reporter gene, whereby a change in the signal from thereporter gene in the presence of the agent compared to the signal fromthe reporter gene in the absence of the agent indicates the agentmodulates MISRII-mediated signal transduction. In some embodiments,where the change is an increase in the signal in step (c) indicates theagent activates and/or increases MISRII mediated signal transduction,whereas where the change is a decrease in the signal in step (c)indicates the agent inhibits and/or decreases MISRII mediated signaltransduction. In some embodiments, the method to identify an agent thatmodulates MISRII-mediated signal transduction in a cell furthercomprises introducing a nucleic acid construct into the cell, where thenucleic acid sequence encodes MISRII or a homologue or fragment thereof,wherein the nucleic acid sequence encoding MISRII is operatively linkedto a promoter, such as, for example a constitutive or inducible ortissue-specific promoter.

In some embodiments, the BMP-responsive element used in the method toidentify an agent that modulates MISRII-mediated signal transduction isSEQ ID NO: 1 or a functional fragment thereof. In some embodiments, thereporter gene in the method to identify an agent that modulatesMISRII-mediated signal transduction encodes a protein with fluorescentand/or chromogenic activity or variants or functional fragments thereof,for example, a chromogenic protein can be a bioluminescent protein orfunctional variants thereof, such as but not limited to a luciferase orfunctional fragments or modified functional versions thereof.

In some embodiments, an agent used to identify an agent that modulatesMISRII-mediated signal transduction is a small molecule, nucleic acid,nucleic acid analogue, aptamer, ribosome, peptide, protein, antibody, orvariants and functional fragments thereof.

In some embodiments, an antibody can be, for example but not limited to,a recombinant antibody, humanized antibody, chimeric antibody, modifiedantibody, monoclonal antibody, polyclonal antibody, miniantibody,dimeric miniantibody, minibody, diabody or tribody or functionalvariants, functional analogues or functional modified versions thereof.In some embodiments, a nucleic acid is DNA, RNA, nucleic acid analogue,peptide nucleic acid (PNA), pseudo-complementary PNA (pcPNA), lockednucleic acid (LNA) or functional analogues thereof, where an RNA can be,for example but not limited to, a small inhibitory RNA, siRNA, microRNA,shRNA, miRNA and functional analogues and homologues and functionalvariants thereof.

Prior to administration, the pyrazoloathrone and functional derivativesthereof as disclosed herein can be formulated as a pharmaceuticalcomposition which contains an effective dosage amount of one or morepyrazoloathrone and functional derivatives thereof in combination withone (or more) pharmaceutically acceptable carrier(s). Conditions thatmay be treated with the pyrazoloathrone and functional derivativesthereof of this invention, or a pharmaceutical composition containingthe same and addition of other therapeutic agents, include any conditionwhich may benefit from administration of MIS, and are particularlyuseful for the prevention and/or treatment of various diseases, forexample proliferative diseases, for example cancers expressing MISreceptors, for example cancer expressing MISRII. Examples of suchcancers are for example, but not limited to, ovarian cancer, vulvarepidermal carcinoma, cervical carcinoma, endometrial edenocarinaoma andovarian adenocarcinoma. In alternative embodiments, the cancerexpressing MIS receptors, for example cancers expressing MISRII are, forexample but not limited to, breast cancer, lung cancer, head and neckcancer, bladder cancer, stomach cancer, cancer of the nervous system,bone cancer, bone marrow cancer, brain cancer, colon cancer, esophagealcancer, endometrial cancer, gastrointestinal cancer, genital-urinarycancer, stomach cancer, lymphomas, melanoma, glioma, bladder cancer,pancreatic cancer, gum cancer, kidney cancer, retinal cancer, livercancer, nasopharynx cancer, ovarian cancer, oral cancers, bladdercancer, hematological neoplasms, follicular lymphoma, cervical cancer,multiple myeloma, osteosarcomas, thyroid cancer, prostate cancer, coloncancer, prostate cancer, skin cancer, stomach cancer, testis cancer,tongue cancer, or uterine cancer.

Another aspect of the present invention relates to the use of apyrazoloanthrone or derivative or analogue thereof for the manufactureof a medicament for treating cancer, wherein the cancer expresses aMullerian Inhibiting Substance (MIS) receptor. In such embodiments, apyrazoloanthrone or functional derivative or functional analogue thereofis anthra[1,9-cd]pyrazol-6(2H)-one or a functional derivative orfunctional analogue thereof. In some embodiments, a cancer cellexpresses a MIS type II receptor or a homologue or functional fragmentthereof.

Another aspect of the present invention relates to an article ofmanufacture comprising packaging material and a pharmaceuticalcomposition of claim 48, wherein the packaging material comprises alabel which indicates the pharmaceutical composition may beadministered, for a sufficient term at an effective dose, for treatingor reducing the risk of cancer which expresses a Mullerian InhibitingSubstance (MIS) receptor.

Another aspect of the present invention relates to a method of treatinga subject affected with cancer, the method comprising assessing theexpression and/or activity of Mullerian Inhibiting Substance Receptor II(MISRII) in a biological sample obtained from the subject, wherein aclinician reviews the results and if the results indicate the presenceof expression and/or activity of MISRII, the clinician directs thesubject to be treated with pharmaceutical composition as disclosedherein. In some embodiments, a biological sample obtained from thesubject is a tissue sample, for example a cancer or tumor tissue sampleor a cancer cell or tumor cell, such as a biopsy tissue sample. In someembodiments, the cancer tissue sample comprises an ovarian cancer cell,a vulvar epidermal carcinoma cell, a cervical carcinoma cell, anendometrial edenocarinaoma cell and/or an ovarian adenocarcinoma cell.In some embodiments, the cancer tissue sample is from a cancer, such as,but not limited to, breast cancer, lung cancer, head and neck cancer,bladder cancer, stomach cancer, cancer of the nervous system, bonecancer, bone marrow cancer, brain cancer, colon cancer, esophagealcancer, endometrial cancer, gastrointestinal cancer, gum cancer, kidneycancer, liver cancer, nasopharynx cancer, ovarian cancer, prostatecancer, skin cancer, stomach cancer, testis cancer, tongue cancer, oruterine cancer.

Another aspect of the present invention relates to the use of apyrazoloanthrone or derivative or analogue thereof to decrease theplasma serum levels of one or more androgens, such as but not limitedto, testosterone, in a subject in need thereof. In some embodiments, thepyrazoloanthrone or derivative or analogue thereof isanthra[1,9-cd]pyrazol-6(2H)-one or a derivative or analogue thereof.

In some embodiments, the subject in need of the pyrazoloanthrone orderivative or analogue thereof has benign prostic hypertrophy. Inalternative embodiments, the subject has prostate cancer, and in someembodiments, the subject has ovarian disease and/or precocious puberty.In alternative embodiments, a subject in need of the pyrazoloanthrone orderivative or analogue thereof can have a disease or disorder selectedfrom the group consisting of; BPH, prostate carcinoma, testicularcancer, androgen dependent acne, male pattern baldness, precociouspuberty, hyperandrogenism, hirsutism, virilization, POCS, HIAR-ANsyndrome, ovarian hyperthecosis, follicular maturation arrest, atresia,anovulation, dysmenorrheal, dysfunctional uterine bleeding, infertilityand androgen-producing tumors.

Another embodiment of the present invention relates to methods to treata disease or disorder characterized by androgenic dependency, comprisingadministering to a subject an effective amount of the pharmaceuticalcomposition comprising a pyrazoloanthrone or derivative or analogue asdisclosed herein, for example SP600125, wherein the pharmaceuticalcomposition reduces the level of at least one androgen in the plasmaserum of the subject and results in a decrease in at least one symptomof a disease or disorder characterized by androgenic dependency.

Another embodiment of the present invention relates to methods todecrease the plasma level of one or more androgens in a subject, themethod comprising administering an effective amount of apyrazoloanthrone or derivative or analogue as disclosed herein, forexample but not limited to, SP600125, wherein the pyrazoloanthrone orderivative or analogue thereof decreases the plasma serum levels of oneor more androgens in the subject. In some embodiments, thepyrazoloanthrone or derivative or analogue thereof isanthra[1,9-cd]pyrazol-6(2H)-one or a derivative or analogue thereof asdisclosed herein. In some embodiments, the subject has a disease ordisorder characterized by androgenic dependency, for example but nolimited to BPH, prostate carcinoma, benign prostic hypertrophy,testicular cancer, androgen dependent acne, male pattern baldness,precocious puberty, hyperandrogenism, hirsutism, virilization, POCS,HIAR-AN syndrome, ovarian hyperthecosis, follicular maturation arrest,atresia, anovulation, dysmenorrheal, dysfunctional uterine bleeding,infertility and androgen-producing tumors.

These and other aspects of this invention will be apparent uponreference to the following detailed description. To that end, certainpatent and other documents are cited herein to more specifically setforth various aspects of this invention. Each of these documents arehereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows SP600125 induces MISRII dependent BRE-luciferase reporterexpression in COS7 cells and increases the MIS response in these cells.COS7 cells were transiently transfected with MISRII, BRE-Luciferase andphRL-CMV. The cells were treated with increasing doses of SP600125 for24 h. Control cells were transfected with a control plasmid (EGFP-N1plasmid) instead of the receptor plasmid to keep the total DNAtransfected constant. The results shown are the fold-induction overvehicle treated cells and normalized to phRL-CMV expression to correctfor variations in cell number and transfection efficiency. Bars shown tothe right of the dashed line indicate that the expression of Renillaluciferase was lower, demonstrating that toxic effects to the cells. Theresults represent the mean values from three independent experimentsperformed in triplicates (Error bars represent SEM, * p<0.05, **p<0.001).

FIGS. 2A-2E shows that SP600125 exerts a synergistic effect onBRE-Luciferase induction in combination with MIS. FIG. 2A shows COS7cells were treated with MIS in combination with increasing doses ofSP600125 or JNK inhibitor III and BRE-Luciferase response was measuredafter 24 h treatment. The results represent the mean values from threeindependent experiments performed in triplicates (Error bars representSEM). * P<0.05, ** P<0.01. FIG. 2B shows transiently transfected COS7cells that were treated with increasing doses of JNK Inhibitor VIII withor without MIS. The results represent the mean values from threedifferent independent experiments. FIG. 2C shows COS7 cells transientlytransfected with MISRII, BRE-Luciferase and phRLCMV were treated with0.5 μg/ml and/or 10 μM SP600125 for 1, 6, 24 and 48 h. Luciferaseactivity was measured and normalized to phRL-CMV activity. The resultsshow the relative light units (RLU) and represent the mean values fromthree independent experiments. a, p<0.05 MIS+SP600125 vs. DMSO, b,p<0.05 MIS and SP600125 vs. DMSO, p<0.01 MIS+SP600125 vs. MIS alone, c,p<0.05 MIS vs. DMSO, P<0.01 MIS+SP600125 vs. MIS. FIG. 2D shows COS7cells that were transfected with either MIS or BMP type II receptortogether with BRE-Luciferase reporter and treated with the respectiveligand with or without MIS for 24 h. Results shown represent the meanvalues form three independent experiments. FIG. 2E shows MOVCAR7 cellstransfected with the BRE-luciferase and phRL-CMV were treated for 24 hafter which the cells were lysed and luciferase activity was measuredand normalized to phRL-CMV. The figure shows the fold induction of BREover un-treated cells. The results shown are the mean values from threeindependent experiments. (Error bars represent SEM, ***p<0.001).

FIG. 3A-3B shows MIS does not activate the JNK-pathway. FIG. 3A showsMOVCAR7 cells treated with either 5 μg/ml MIS or 500 nM Anisomycin for30 min before harvesting and subjecting the lysates to immunoblotsrecognizing the phosphorylated forms of JNK. FIG. 3B showsphosphorylation of c-jun induced by 500 nM Anisomycin. Cells werepre-treated with the indicated doses of SP600125 induced activation ofph-c-jun was inhibited by 25 μM SP600125 or JNK inhibitor VIII (25 μM).The results are representative of several experiments.

FIG. 4A-4C shows SP600125 inhibits proliferation of MOVCAR7 cells andincreases the efficiency of paclitaxel-mediated inhibition ofproliferation as detected in an MTT-assay. Cells were plated in 96-wellplates and treated the day after. Seven days after plating the cellsurvival was measured using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide substrateand measuring the absorbance at 550 nm. FIG. 4A shows the effect ofSP600125 on cell proliferation and survival by comparing cell-linesexpressing MISRII (MOVCAR7 cells), and COS7 cells that do not expressMISRII. FIG. 4B shows increasing doses of a known ovarian cancer drug,paclitaxel (PTX), was used in combination with SP600125. FIG. 4C showsthe effect of 2 μM SP600125, 10 μg/MIS, and 3 nM paclitaxel alone or incombination on another MIS-responsive mouse ovarian cancer cel line,4306. The results shown are the mean values from three independentexperiments. (Error bars represent SEM, ***p<0.001). Significantdifferences at SP600125 concentrations between each treatment group (inFIG. 4B) were determined by two-way factorial ANOVA, ***, P<0.001.Significant differences (P<0.05) in FIG. 4C were determined by one-wayfactorial ANOVA. If any give pair of bars shares the same letter, it isnot significantly different.

FIG. 5 shows SP600125 induces MISRII-dependent BRE-luciferase reporterexpression in COS7 cells and increase MIS response to these cells. COS7cells were transfected with normal MISRII or a kinase-deficient receptorMISRII K228R and treated with increasing doses of SP600125 and/or MISfor 24 hrs. Results shown represent the mean values from fourindependent experiments performed in triplicates (Error bars representstandard error of the mean, *, P<0.05). These results demonstrate thatthe kinase activity of MISRII is required to observe SP600125 mediatedactivation of the MIS signal transduction pathway.

DETAILED DESCRIPTION

As discussed herein, the present invention provides a method fortreating a variety of conditions by administering an effective amount ofa pyrazoloathrone and functional derivatives thereof of the invention toa subject in need thereof. Conditions that may be treated by thecompounds of this invention, or a pharmaceutical composition containingthe same, include any condition which is treated or reduces the symptomsby administration of MIS or activation of MIS signaling or activation ofMISRII, and thereby benefit from administration of a pyrazoloathrone andfunctional derivatives thereof. Representative conditions in this regardinclude, for example, but not limited to, cancers that express MISreceptors, for example cancer that express MISRII, for example, but notlimited to ovarian, cervical and endometrial cancer. Other conditionswhich can be treated with MIS or activation of MIS signalling reducesthe symptoms are, for example, rheumatoid arthritis, proliferativediseases such as cancer, treatment of prostatic cancer, polycysicovarian disease, benign prostatic hypertrophy and precocious puberty andother hyperandrogen disorders such as testitoxicosis.

DEFINITIONS

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

The term “Mullerian Inhibiting Substance” and “MIS” are usedinterchangeably herein and is also known as anti-Müllerian hormone orAMH, refer to compounds and materials which are structurally similar toMIS. Examples of such intended substances are for example, salts,functional derivatives and aglycone forms of MIS. Additionally, thepresent invention is intended to include mutant forms of MIS which havesubstantially the same biological activity as MIS. Examples of suchmutant MIS molecules carrying a deletion, insertion, or alteration inamino acid sequence. MIS can be obtained from any mammalian source orfrom non-mammalian sources through the use of recombinant DNAtechnology, or from chemical synthesis of the MIS protein. For referencepurposes only, the human MIS nucleic acid corresponds to ReSeq No:NM_(—)000479 (SEQ ID NO:2) and GenBank No: KO3474 (SEQ ID NO:3), whichare incorporated herein by reference.

The term “Mullerian Inhibiting Substance type II receptor” or “MISRII”are used interchangeably herein refer to the type II receptor for MIS.The term MISRII is intended to encompass all MIS receptors substantiallyhomologous to MISRII and functional derivatives of MISRII. MISRII isalso known by the alias as AMHR2, and for reference purposes, thenucleic acid sequence of human MISRII corresponds to NM_(—)020547 (SEQID NO:4) and GenBank No: AF172932 (SEQ ID NO:5) which are incorporatedherein by reference

The term “functional derivative” and “mimetic” are used interchangeablyherein, and refers to compounds which possess a biological activity (inparticular functional biological activity) that is substantially similarto the biological activity of the entity or molecule for which it's afunctional derivative of. The term functional derivative is intended toinclude the fragments, variants, analogues or chemical derivatives of amolecule. In certain embodiments, functional derivatives and functionalanalogues of pyrazoloathrones can be assessed for their biologicalactivity using the assay as disclosed herein, where derivatives andanalogues which activate MIS RII signalling using the assay as disclosedin the Examples would be considered as functional derivatives orfunctional analogues of pyrazoloathrone.

The term “analog” as used herein refers to an agent that retains thesame, or a substantially similar biological function (i.e., binding to areceptor, such as MIS RII) and/or structure as the molecule or chemicalor polypeptide it is an analogue of. Examples of analogs includepeptidomimetics (a peptide analog), peptide nucleic acids (a nucleicacid analog), small and large organic or inorganic compounds, as well asderivatives and variants of a polypeptide or nucleic acid herein.

The term “substantially similar”, when used to define the biologicalactivity of a derivative or analogue of pyrazoloathrone as compared tothe biological activity of the pyrazoloathrone to which it is aderivative or analogue of, means that a particular derivative oranalogue differs from the initial pyrazoloathrone in chemical structure,by one or more groups or elements, including substitutions, deletions,or additions of groups of elements, the net effect of which is to retainat least some of the biological activity found in the initialpyrazoloathrone with respect to the biological activity ofpyrazoloathrone with respect to activation of the MISRII signallingpathyway. Such biological activity can be assessed by one of ordinaryskill in the art using the assay as disclosed herein. As such,derivative or analogue of pyrazoloathrones having lesser degrees ofstructural similarity but a substantially similar or comparablebiological activity of the original pyrazoloathrone from which is basedwith respect to activation of MISRII signalling are considered to beequivalents. Substantially similar derivatives or analogues ofpyrazoloathrone will typically have at least about 60%, or at leastabout 70% or at least about 80% or at least about 90% or at least about95%, or at least about 100% the biological activity of MISRII activationas compared to the pyrazoloathrone it is a derivative or analogue of, orat least at least 2-fold, or at least about 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold, or anyincrease between 2-fold and 10-fold or greater the biological activityof MISRII activation as compared to the pyrazoloathrone are to beconsidered a functional derivative or a functional analogue of thepyrazoloathrone they are based on, as can be assayed using the methodsas disclosed herein.

The terms “lower”, “reduced”, “reduction” or “decrease” or “inhibit” areall used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “lower”, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(i.e. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

As used herein, the term “subject” refers to any living organism whichcan be administered to the pharmaceutical compositions of the presentinvention and in which cancer or a proliferative disorder can occur. Theterm includes, but is not limited to, humans, non-human primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses, domestic subjects such as dogsand cats, laboratory animals including rodents such as mice, rats andguinea pigs, and the like. The term does not denote a particular age orsex. Thus, adult and newborn subjects, as well as fetuses, whether maleor female, are intended to be covered. The term “subject” is alsointended to include living organisms susceptible to conditions ordisease states as generally disclosed, but not limited to, throughoutthis specification. Examples of subjects include humans, dogs, cats,cows, goats, and mice. The term subject is further intended to includetransgenic species. As used herein, the terms “subject” and “individual”are used interchangeably and are intended to refer to an animal, forexample a human, to whom treatment, including prophylactic treatment,with the pharmaceutical composition according to the present invention,is provided, including, but not limited to humans and non-human animals.The term “non-human animals” and “non-human mammals” are usedinterchangeably herein includes all vertebrates, e.g., mammals, such asnon-human primates, (particularly higher primates), sheep, dog, rodent(e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, andnon-mammals such as chickens, amphibians, reptiles etc. In oneembodiment, the subject is human. In another embodiment, the subject isan experimental animal or animal substitute as a disease model.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

The term “disease” or “disorder” is used interchangeably herein, refersto any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,amlady, disorder, sickness, illness, complaint, inderdisposion,affection.

The term “malignancy” and “cancer” are used interchangeably herein,refers to diseases that are characterized by uncontrolled, abnormalgrowth of cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. The term isalso intended to include any disease of an organ or tissue in mammalscharacterized by poorly controlled or uncontrolled multiplication ofnormal or abnormal cells in that tissue and its effect on the body as awhole. Cancer diseases within the scope of the definition comprisebenign neoplasms, dysplasias, hyperplasias as well as neoplasms showingmetastatic growth or any other transformations like e.g. leukoplakiaswhich often precede a breakout of cancer.

The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, to whom treatment,including prophylactic treatment, with a pharmaceutical composition asdisclosed herein, is provided. The term “subject” as used herein refersto human and non-human animals. The term “non-human animals” and“non-human mammals” are used interchangeably herein and includes allvertebrates, e.g., mammals, such as non-human primates, (particularlyhigher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig,goat, pig, cat, rabbits, cows, and non-mammals such as chickens,amphibians, reptiles etc. In one embodiment, the subject is human. Inanother embodiment, the subject is an experimental animal or animalsubstitute as a disease model.

As used herein, the term “tumor” refers to a mass of transformed cellsthat are characterized, at least in part, by containing angiogenicvasculature. The transformed cells are characterized by neoplasticuncontrolled cell multiplication which is rapid and continues even afterthe stimuli that initiated the new growth has ceased. The term “tumor”is used broadly to include the tumor parenchymal cells as well as thesupporting stroma, including the angiogenic blood vessels thatinfiltrate the tumor parenchymal cell mass. Although a tumor generallyis a malignant tumor, i.e., a cancer having the ability to metastasize(i.e. a metastatic tumor), a tumor also can be nonmalignant (i.e.non-metastatic tumor). Tumors are hallmarks of cancer, a neoplasticdisease the natural course of which is fatal. Cancer cells exhibit theproperties of invasion and metastasis and are highly anaplastic.

As used herein, the term “metastases” or “metastatic tumor” refers to asecondary tumor that grows separately elsewhere in the body from theprimary tumor and has arisen from detached, transported cells, whereinthe primary tumor is a solid tumor. The primary tumor, as used herein,refers to a tumor that originated in the location or organ in which itis present and did not metastasize to that location from anotherlocation. As used herein, a “malignant tumor” is one having theproperties of invasion and metastasis and showing a high degree ofanaplasia. Anaplasia is the reversion of cells to an immature or a lessdifferentiated form, and it occurs in most malignant tumors.

The term “therapy resistant cancer” as used herein refers to a cancerpresent in a subject which is resistant to, or refractory to at leasttwo different anti-cancer agents such as chemotherapy agents, whichmeans, typically a subject has been treated with at least two differentanti-cancer agents that did not provide effective treatment as that termis defined herein.

The term ‘sensitize’ or ‘sensitizes’ used interchangeably herein, refersto making the cell sensitive, or susceptible to other secondary agents,for example other pro-drugs or other environmental effects such asradiation etc.

The term “cell” used herein refers to any cell, prokaryotic oreukaryotic, including plant, yeast, worm, insect and mammalian.Mammalian cells include, without limitation; primate, human and a cellfrom any animal of interest, including without limitation; mouse,hamster, rabbit, dog, cat, transgenic animal domestic animals, such asequine, bovine, murine, ovine, canine, feline, etc. The cells may be awide variety of tissue types without limitation such as; hematopoietic,neural, mesenchymal, cutaneous, mucosal, stromal, muscle spleen,reticuloendothelial, epithelial, endothelial, hepatic, kidney,gastrointestinal, pulmonary, T-cells etc. Stem cells, embryonic stem(ES) cells, ES-derived cells and stem cell progenitors are alsoincluded, including without limitation, hematopoeitic, stromal, muscle,cardiovascular, hepatic, pulmonary, gastrointestinal stem cells, etc.Yeast cells may also be used as cells in this invention. Cells alsorefer not to a particular subject cell but to the progeny or potentialprogeny of such a cell because of certain modifications or environmentalinfluences, for example differentiation, such that the progeny may not,in fact be identical to the parent cell, but are still included in thescope of the invention.

The cells used in the invention can also be cultured cells, e.g. invitro or ex vivo. For example, cells cultured in vitro in a culturemedium. Alternatively, for ex vivo cultured cells, cells can be obtainedfrom a subject, where the subject is healthy and/or affected with adisease. Cells can be obtained, as a non-limiting example, by biopsy orother surgical means know to those skilled in the art. Cells used in theinvention can be present in a subject, e.g. in vivo. For the inventionon use on in vivo cells, the cell is preferably found in a subject anddisplay characteristics of the disease, disorder, or malignancypathology

As used herein, the terms “treat” or “treatment” or “treating” refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow the development of the disease,such as slow down the development of a tumor, the spread of cancer, orreducing at least one effect or symptom of a condition, disease ordisorder associated with inappropriate proliferation or a cell mass, forexample cancer. Treatment is generally “effective” if one or moresymptoms or clinical markers are reduced as that term is defined herein.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of at leastslowing of progress or worsening of symptoms that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already diagnosedwith cancer, as well as those likely to develop secondary tumors due tometastasis.

The term “effective amount” as used herein refers to the amount oftherapeutic agent of pharmaceutical composition, such as thepharmaceutical composition comprising at least one pyrazoloanthrones asdisclosed herein, to alleviate at least one or more symptom of thedisease or disorder, and relates to a sufficient amount ofpharmacological composition to provide the desired effect. The phrase“therapeutically effective amount” as used herein, e.g., apharmaceutical composition comprising at least one pyrazoloanthrones asdisclosed herein means a sufficient amount of the composition to treat adisorder, at a reasonable benefit/risk ratio applicable to any medicaltreatment. The term “therapeutically effective amount” therefore refersto an amount of the composition as disclosed herein that is sufficientto effect a therapeutically or prophylacticly significant reduction in asymptom or clinical marker associated with a cancer or a cancer-mediatedcondition.

A therapeutically or prophylatically significant reduction in a symptomis, e.g. at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 125%, at least about 150% or more in a measured parameter ascompared to a control or non-treated subject. Measured or measurableparameters include clinically detectable markers of disease, forexample, elevated or depressed levels of a biological marker, as well asparameters related to a clinically accepted scale of symptoms or markersfor a disease or disorder. It will be understood, however, that thetotal daily usage of the compositions and formulations as disclosedherein will be decided by the attending physician within the scope ofsound medical judgment. The exact amount required will vary depending onfactors such as the type of disease being treated.

With reference to the treatment of a subject with a cancer with apharmaceutical composition comprising at least one pyrazoloanthrones asdisclosed herein, the term “therapeutically effective amount” refers tothe amount that is safe and sufficient to prevent or delay thedevelopment and further growth of a tumor or the spread of metastases incancer patients. The amount can thus cure or cause the cancer to go intoremission, slow the course of cancer progression, slow or inhibit tumorgrowth, slow or inhibit tumor metastasis, slow or inhibit theestablishment of secondary tumors at metastatic sites, or inhibit theformation of new tumor metastases. The effective amount for thetreatment of cancer depends on the tumor to be treated, the severity ofthe tumor, the drug resistance level of the tumor, the species beingtreated, the age and general condition of the subject, the mode ofadministration and so forth. Thus, it is not possible to specify theexact “effective amount”. However, for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing only routine experimentation. The efficacy of treatment can bejudged by an ordinarily skilled practitioner, for example, efficacy canbe assessed in animal models of cancer and tumor, for example treatmentof a rodent with a cancer, and any treatment or administration of thecompositions or formulations that leads to a decrease of at least onesymptom of the cancer, for example a reduction in the size of the tumoror a slowing or cessation of the rate of growth of the tumor indicateseffective treatment. In embodiments where the compositions are used forthe treatment of cancer, the efficacy of the composition can be judgedusing an experimental animal model of cancer, e.g., wild-type mice orrats, or preferably, transplantation of tumor cells. When using anexperimental animal model, efficacy of treatment is evidenced when areduction in a symptom of the cancer, for example a reduction in thesize of the tumor or a slowing or cessation of the rate of growth of thetumor occurs earlier in treated, versus untreated animals. By “earlier”is meant that a decrease, for example in the size of the tumor occurs atleast 5% earlier, but preferably more, e.g., one day earlier, two daysearlier, 3 days earlier, or more.

As used herein, the term “treating” when used in reference to a cancertreatment is used to refer to the reduction of a symptom and/or abiochemical marker of cancer, for example a reduction in at least onebiochemical marker of cancer by at least about 10% would be consideredan effective treatment. Examples of such biochemical markers of cancerinclude CD44, telomerase, TGF-α, TGF-β, erbB-2, erbB-3, MUC1, MUC2,CK20, PSA, CA125 and FOBT. A reduction in the rate of proliferation ofthe cancer cells by at least about 10% would also be consideredeffective treatment by the methods as disclosed herein. As alternativeexamples, a reduction in a symptom of cancer, for example, a slowing ofthe rate of growth of the cancer by at least about 10% or a cessation ofthe increase in tumor size, or a reduction in the size of a tumor by atleast about 10% or a reduction in the tumor spread (i.e. tumormetastasis) by at least about 10% would also be considered as affectivetreatments by the methods as disclosed herein. In some embodiments, itis preferred, but not required that the therapeutic agent actually killthe tumor.

As used herein, the terms “administering,” and “introducing” are usedinterchangeably herein and refer to the placement of the pharmaceuticalcompositions of the present invention comprising the pyrazoloanthronesof the present invention into a subject by a method or route whichresults in at least partial localization of the pyrazoloanthrones at adesired site. The compounds of the present invention can be administeredby any appropriate route which results in an effective treatment in thesubject.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal,intracerebrospinal, and intrasternal injection and infusion. The phrases“systemic administration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein mean theadministration of the pharmaceutical compositions of the presentinvention comprising pyrazoloanthrones and optionally other agents ormaterial other than directly into the central nervous system, such thatit enters the animal's system and, thus, is subject to metabolism andother like processes, for example, subcutaneous administration.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in maintaining the activity of or carrying ortransporting the subject agents from one organ, or portion of the body,to another organ, or portion of the body. In addition to being“pharmaceutically acceptable” as that term is defined herein, eachcarrier must also be “acceptable” in the sense of being compatible withthe other ingredients of the formulation. The pharmaceutical formulationcontains a compound of the invention in combination with one or morepharmaceutically acceptable ingredients. The carrier can be in the formof a solid, semi-solid or liquid diluent, cream or a capsule. Thesepharmaceutical preparations are a further object of the invention.Usually the amount of active compounds is between 0.1-95% by weight ofthe preparation, preferably between 0.2-20% by weight in preparationsfor parenteral use and preferably between 1 and 50% by weight inpreparations for oral administration. For the clinical use of themethods of the present invention, targeted delivery composition of theinvention is formulated into pharmaceutical compositions orpharmaceutical formulations for parenteral administration, e.g.,intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical,e.g., transdermal; ocular, e.g., via corneal scarification or other modeof administration. The pharmaceutical composition contains a compound ofthe invention in combination with one or more pharmaceuticallyacceptable ingredients. The carrier can be in the form of a solid,semi-solid or liquid diluent, cream or a capsule.

The terms “composition” or “pharmaceutical composition” usedinterchangeably herein refer to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. Suchcompositions can be specifically formulated for administration via oneor more of a number of routes, including but not limited to, oral,ocular parenteral, intravenous, intraarterial, subcutaneous, intranasal,sublingual, intraspinal, intracerebroventricular, and the like. Inaddition, compositions for topical (e.g., oral mucosa, respiratorymucosa) and/or oral administration can form solutions, suspensions,tablets, pills, capsules, sustained-release formulations, oral rinses,or powders, as known in the art are described herein. The compositionsalso can include stabilizers and preservatives. For examples ofcarriers, stabilizers and adjuvants, University of the Sciences inPhiladelphia (2005) Remington: The Science and Practice of Pharmacy withFacts and Comparisons, 21st Ed.

The term “agent” or “compound” as used herein refers to a chemicalentity or biological product, or combination of chemical entities orbiological products, administered to a subject to treat or prevent orcontrol a disease or condition. The chemical entity or biologicalproduct is preferably, but not necessarily a low molecular weightcompound, but may also be a larger compound, or any organic or inorganicmolecule, including modified and unmodified nucleic acids such asantisense nucleic acids, RNAi, such as siRNA or shRNA, peptides,peptidomimetics, receptors, ligands, and antibodies, aptamers,polypeptides, nucleic acid analogues or variants thereof. For example,an oligomer of nucleic acids, amino acids, or carbohydrates includingwithout limitation proteins, oligonucleotides, ribozymes, DNAzymes,glycoproteins, siRNAs, lipoproteins, aptamers, and modifications andcombinations thereof.

The term “vectors” used interchangeably with “plasmid” refer to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. Vectors capable of directing the expression ofgenes and/or nucleic acid sequence to which they are operatively linkedare referred to herein as “expression vectors”. In general, expressionvectors of utility in recombinant DNA techniques are often in the formof “plasmids” which refer to circular double stranded DNA loops which,in their vector form are not bound to the chromosome. Other expressionvectors can be used in different embodiments of the invention, forexample, but are not limited to, plasmids, episomes, bacteriophages orviral vectors, and such vectors may integrate into the host's genome orreplicate autonomously in the particular cell. Other forms of expressionvectors known by those skilled in the art which serve the equivalentfunctions can also be used. Expression vectors comprise expressionvectors for stable or transient expression encoding the DNA.

As used herein, a “promoter” or “promoter region” or “promoter element”used interchangeably herein refers to a segment of a nucleic acidsequence, typically but not limited to DNA or RNA or analogues thereof,that controls the transcription of the nucleic acid sequence to which itis operatively linked. The promoter region includes specific sequencesthat are sufficient for RNA polymerase recognition, binding andtranscription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences which modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis-actingor may be responsive to trans-acting factors. Promoters, depending uponthe nature of the regulation may be constitutive or regulated.

The term “regulatory sequences” is used interchangeably with “regulatoryelements” herein refers element to a segment of nucleic acid, typicallybut not limited to DNA or RNA or analogues thereof, that modulates thetranscription of the nucleic acid sequence to which it is operativelylinked, and thus act as transcriptional modulators. Regulatory sequencesmodulate the expression of gene and/or nucleic acid sequence to whichthey are operatively linked. Regulatory sequence often comprise“regulatory elements” which are nucleic acid sequences that aretranscription binding domains and are recognized by the nucleicacid-binding domains of transcriptional proteins and/or transcriptionfactors, repressors or enhancers etc. Typical regulatory sequencesinclude, but are not limited to, transcriptional promoters, an optionaloperate sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences to control the terminationof transcription and/or translation. Regulatory sequences are selectedfor the assay to control the expression of split-biomolecular conjugatein a cell-type in which expression is intended.

Regulatory sequences can be a single regulatory sequence or multipleregulatory sequences, or modified regulatory sequences or fragmentsthereof. Modified regulatory sequences are regulatory sequences wherethe nucleic acid sequence has been changed or modified by some means,for example, but not limited to, mutation, methylation etc.

The term “operatively linked” or “operatively associated” are usedinterchangeably herein, and refer to the functional relationship of thenucleic acid sequences with regulatory sequences of nucleotides, such aspromoters, enhancers, transcriptional and translational stop sites, andother signal sequences. For example, operative linkage of nucleic acidsequences, typically DNA, to a regulatory sequence or promoter regionrefers to the physical and functional relationship between the DNA andthe regulatory sequence or promoter such that the transcription of suchDNA is initiated from the regulatory sequence or promoter, by an RNApolymerase that specifically recognizes, binds and transcribes the DNA.In order to optimize expression and/or in vitro transcription, it may benecessary to modify the regulatory sequence for the expression of thenucleic acid or DNA in the cell type for which it is expressed. Thedesirability of, or need of, such modification may be empiricallydetermined.

The term “oncogene” as used herein refers to a nucleic acid sequenceencoding, or polypeptide, of a mutated and/or overexpressed version of anormal gene that in a dominant fashion can release the cell from normalrestraints on growth and thus alone or in concert with other changes,contribute to a cells tumorigenicity. Examples of oncogenes include;gp40 (v-fms); p21 (ras); p55 (v-myc); p65 (gag-jun); pp60 (v-src);v-abl; v-erb; v-erba; v-fos etc. A proto-oncogene refers to the normalexpression of a nucleic acid expressing the normal, cellular equivalentof an oncogene, typically these genes are usually a gene involved in thesignaling or regulation of cell growth.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%. The present invention is further explained in detail by thefollowing examples, but the scope of the invention should not be limitedthereto.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Compositions of Pyrazoloathrone and Derivatives

As mentioned above, the present invention is directed to compounds whichhave activity as selective inhibitors of JNK, as well as to compositionsand methods relating to the

same. The compounds of this invention have the following structure (I),also known herein as Compound (II):and pharmaceutically acceptable salts thereof, wherein:

R₁ and R₂ are optional substituents that are the same or different andindependently represent alkyl, halogen, nitro, trifluoromethyl,sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy,arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,aminoalkoxy, mono- or di-alkylaminoalkoxy, or a group represented byformula (a), (b), (c) or (d):

R₃ and R₄ taken together represent alkylidene or a heteroatom-containingalkylidene, or R₃ and R₄ are the same or different and independentlyrepresent hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl,aryloxyalkyl, alkoxyalkyl, alkoxyamino, or alkoxy(mono- ordi-alkylamino); and

R₅ represents hydrogen, alkyl, cycloalkyl, aryl, arylalkyl,cycloalkylalkyl, alkoxy, amino, mono- or di-alkylamino, arylamino,arylalkylamino, cycloalkylamino, or cycloalkylalkylamino.

As used herein, the terms used above having following meaning.

“Alkyl” means a straight chain or branched, saturated or unsaturatedalkyl chain having from 1 to 8 carbon atoms, such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, propylenyl,1-butenyl, propynyl, and the like.

“Halogen” means fluorine, chlorine, bromine or iodine.

“Trifluoromethyl” means —CF₃.

“Sulfonyl” means —SO₃H.

“Carboxyl” means —COOH.

“Alkoxy” means —O-(alkyl), such as methoxy, ethoxy, n-propyloxy,iso-propyloxy, n-butyloxy, iso-butyloxy, and the like.

“Alkoxyalkoxy” means —O-(alkyl)-O-(alkyl), such as —OCH₂CH₂OCH₃, and thelike.

“Alkoxycarbonyl” means —C(|O)O-(alkyl), such as —C(═O)OCH₃,—C(═O)OCH₂CH₃, and the like.

“Alkoxyalkyl” means -(alkyl)-O-(alkyl), such as —CH₂OCH₃, —CH₂OCH₂CH₃,and the like.

“Aryl” means a carbocyclic or heterocyclic aromatic group containingfrom 5 to 10 ring atoms. The ring atoms of a carbocyclic aryl group areall carbon atoms, and includes phenyl and naphthyl. The ring atoms of aheterocyclic aryl group contains at least one heteroatom selected fromnitrogen, oxygen and sulfur, and include pyridinyl, pyrimidinyl,furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl,pyrazinyl, triazinyl, tetrazolyl, and indolyl.

“Aryloxy” means —O-(aryl), such as —O-phenyl, —O-pyridinyl, and thelike.

“Arylalkyl” means -(alkyl)-(aryl), such as benzyl (i.e., —CH₂phenyl),—CH₂-pyrindinyl, and the like.

“Arylalkyloxy” means —O-(alkyl)-(aryl), such as —O-benzyl,—O—CH₂-pyridinyl, and the like.

“Cycloalkyl” means a cyclic alkyl having from 3 to 7 carbon atoms, suchas cyclopropyl, cyclopentyl, cyclohexyl, and the like.

“Cycloalkyloxy” means —O-(cycloalkyl), such as —O-cyclohexyl, and thelike.

“Cycloalkylalkyloxy” means —O-(alkyl)-(cycloalkyl, such as—OCH₂cyclohexyl, and the like.

“Alkylidene” means the divalent radical —CH_(n)H_(2n)—, wherein n is aninteger from 1 to 8, such as —CH₂—, —CH₂CH₂—, —CH₂—CH₂—CH₂—,—CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, and the like.

“Heteroatom-containing alkylidene” means an alkylidene wherein at leastone carbon atom is replaced by a heteroatom selected from nitrogen,oxygen or sulfur, such as —CH₂CH₂OCH₂CH₂—, and the like.

“Aminoalkoxy” means —O-(alkyl)-NH₂, such as —OCH₂NH₂, —OCH₂CH₂NH₂, andthe like.

“Mono- or di-alkylamino” means —NH(alkyl) or —N(alkyl)(alkyl),respectively, such as —NHCH₃, —N(CH₃)₂, and the like.

“Mono- or di-alkylaminoalkoxy” means —O-(alkyl)-NH(alkyl) or—O-(alkyl)-N(alkyl)(alkyl), respectively, such as —OCH₂NHCH₃,—OCH₂CH₂N(CH₃)₂, and the like.

“Arylamino” means —NH(aryl), such as —NH-phenyl, —NH-pyridinyl, and thelike.

“Arylalkylamino” means —NH-(alkyl)-(aryl), such as —NH-benzyl,—NHCH₂-pyridinyl, and the like.

“Alkylamino” means —NH(alkyl), such as —NHCH₃, —NHCH₂CH₃, and the like.

“Cycloalkylamino” means —NH-(cycloalkyl), such as —NH-cyclohexyl, andthe like.

“Cycloalkylalkylamino” —NH-(alkyl)-(cycloalkyl), such as—NHCH₂-cyclohexyl, and the like.

In the embodiment wherein R₁ and R₂ are not present, compounds of thisinvention have the following structure (II) (also referred to herein as“Compound (I)”):

This compound is commercially available from Pfaltz-Bauer (Conn., U.S.)and also StressGen Bioreagents, (MI, U.S), also referred to as SP600125and/or anthra[1,9-cd]pyrazol-6(2H)-one.

In the embodiment wherein only one of R₁ and R₂ is present, compounds ofthis invention have one of the following

In the embodiment wherein both R₁ and R₂ are present, compounds of thisinvention have one of the following structures (V), (VI) or (VII):

The compound Anthra[1,9cd]pyrazol-6(2H)-one (“Compound 1” or “SP600125”)is a yellow solid and is synthesized by the addition of anhydroushydrazine to a solution of 2-chloroanthraquinone (Aldrich) in 10 mlpyridine, and the mixture heated at 100° C. for 16 hours. The mixture iscooled and the solvent is evaporated in vacuo. The residue is taken inhot 6N HCl, and the solid is collected by filtration. Flashchromatography of the crude material on silica gel affordsanthra[1,9cd]pyrazol-6(2H)-one (“Compound 1”) as yellow solids. Forexample, the reaction scheme is:

The above reaction yields Anthra[1,9cd]pyrazol-6(2H)-one (“Compound (I)”or “SP600125”):

Purification of Anthra[1,9cd]pyrazol-6(2H)-one (“Compound (I)” or“SP600125”) can be performed as previously described in the methodsdisclosed in U.S. Pat. No. 7,119,114, which is incorporated herein inits entirety by reference.

Pharmaceutically acceptable salts of compounds of structure (I) orstructure (II) are also within the scope of this invention. To this end,the compound may generally be utilized as the free base. Alternatively,the compounds may be used in the form of acid addition salts. Acidaddition salts of the free base amino compounds of the present inventionmay be prepared by methods well known in the art, and may be formed fromorganic and inorganic acids. Suitable organic acids include maleic,fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, oxalic,propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic,cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, andbenzenesulfonic acids. Suitable inorganic acids include hydrochloric,hydrobromic, sulfuric, phosphoric, and nitric acids. Thus, the term“pharmaceutically acceptable salt” of a compound of structure (I) isintended to encompass any and all acceptable salt forms.

The compounds of the present invention may generally be made by organicsynthesis techniques known to those skilled in the art, as well as bythe methods disclosed in U.S. Pat. No. 7,119,114, which is incorporatedherein in its entirety by reference.

Method of Treatment of a Subject

The present invention relates generally to a method of treating aproliferative disease or disorder in a subject, where the proliferativedisease or disorder is associated with cells that express a MISreceptor, for example cells expressing MISRII. In some embodiments, theproliferative disease or disorder is cancer, where the cancer or cancercells express at least one MIS receptor, for example cancer or cancercells expressing MISRII. The method of the present invention comprisesthe administration of an effective amount of Compound I of the inventionor analogue or derivative thereof to a subject in with a proliferativedisorder, where the cells associated with the proliferative disorderexpress at least one MIS receptors, for example the cells expressMISRII. For example, an effective amount of Compound I of the inventionor analogue or derivative thereof is administered to a subject with acancer expressing at least one MIS receptors, for example expressingMISRII. Thus, by using the methods of the present invention, one canintervene in the proliferative disease, for example cancer, amelioratethe symptoms, and in some cases cure the disease.

Examples of such diseases where proliferation of cells expressing atleast one MIS receptors, for example expressing MISRII is the cause ofdisease are cancers, for example cervical cancer and ovarian cancer. Insome embodiments, the cancer expressing at least one MIS receptor, forexample MISRII is a cancer cell. In some embodiments, such a cancer cellexpressing at least one MIS receptors, for example expressing MISRII is,for example but not limited to, an ovarian cancer cell, vulvar epidermalcarcinoma cell, cervical carcinoma cell, endometrial edenocarinaomacell, ovarian adenocarcinoma.

In alternative embodiments, the cancer expressing at least one MISreceptor, for example cancers expressing MISRII are for example but notlimited to; breast cancer, lung cancer, head and neck cancer, bladdercancer, stomach cancer, cancer of the nervous system, bone cancer, bonemarrow cancer, brain cancer, colon cancer, esophageal cancer,endometrial cancer, gastrointestinal cancer, genital-urinary cancer,stomach cancer, lymphomas, melanoma, glioma, bladder cancer, pancreaticcancer, gum cancer, kidney cancer, retinal cancer, liver cancer,nasopharynx cancer, ovarian cancer, oral cancers, bladder cancer,hematological neoplasms, follicular lymphoma, cervical cancer, multiplemyeloma, osteosarcomas, thyroid cancer, prostate cancer, colon cancer,prostate cancer, skin cancer, stomach cancer, testis cancer, tonguecancer, or uterine cancer.

In alternative embodiments, the present invention relates to the use ofpyrazoloathrone and functional derivatives thereof for the treatment ofany disorder where administration of the MIS protein or a nucleic acidencoding MIS protein or activation MISRII is whole or part of thetherapeutic regime.

In some embodiments, the cancer is a MIS-responsive cancer, for examplebut not limited ovarian cancer and cervical cancer. In some embodiments,the cancer expresses MISRII, for example but not limited ovarian cancerand cervical cancer. In some embodiments, the disorder is a disorderassociated with excess androgen states, for example as disclosed in U.S.Pat. No. 6,673,352, which is incorporated in its entirety herein byreference. In some embodiments, the methods of the present invention areused in the treatment of prostatic cancer, polycysic ovarian disease,benign prostatic hypertrophy and precocious puberty.

In a related embodiment, a tissue to be treated is a tumor tissueexpressing at least one MIS receptor, for example expressing MISRII of asubject, for example the tumor tissue is, but not limited to a solidtumor, a metastases, a skin cancer, a breast cancer, an ovarian cancer,an cervical cancer, a hemangioma or angiofibroma and the like cancer.Typical solid tumor tissues treatable by the pharmaceutical compositionof the invention, includes for example, but not limited to tumors of thelung, pancreas, breast, colon, laryngeal, ovarian, and the like tissues.In some embodiment, the solid tumor tissue treatable by the presentmethods include thyroid, and the cancer type is medullary thyroidcancer.

In a related embodiment, the invention contemplates the practice of themethod in conjunction with other therapies such as conventionalchemotherapy directed against solid tumors and for control ofestablishment of metastases. The administration of the compoundsdescribed herein is typically conducted prior to and/or at the same timeand/or after chemotherapy, although it is also encompassed within thepresent invention to inhibit cell proliferation after a regimen ofchemotherapy at times where the tumor tissue will be responding to thetoxic assault by inducing angiogenesis to recover by the provision of ablood supply and nutrients to the tumor tissue. In addition, thepharmaceutical compositions of the invention for the treatment ofproliferative disorders, for example cancer, can be administratedprophylatically and/or before the development of a tumor, if the subjecthas been identified as to have a risk of developing cancer, for exampleto subjects that are positive for biomarkers of cancer cells or tumors.Insofar as the present methods apply to inhibition of cellproliferation, the methods can also apply to inhibition of tumor tissuegrowth, to inhibition of tumor metastases formation, and to regressionof established tumors.

The presence of MISRII in fluids such as blood may be indicative of thepresence of cancer. The presence of MISRII in fluids or sites not near atumor may be indicative of metastasis. In some such embodiments, thecompounds of the present invention are administered to the subject, andin some embodiments the compounds of the present invention areadministered to the subject in a pharmaceutical composition comprisingone or more additional therapies.

The inventive methods disclosed herein provide for the parenteral andoral administration of the compounds of the present invention, incombination with other pharmaceutical compositions to subjects in needof such treatment. Parenteral administration includes, but is notlimited to, intravenous (IV), intramuscular (IM), subcutaneous (SC),intraperitoneal (IP), intranasal, and inhalant routes. In the method ofthe present invention, the resolvins and/or protectins or analogsthereof are preferably administered orally. IV, IM, SC, and IPadministration may be by bolus or infusion, and may also be by slowrelease implantable device, including, but not limited to pumps, slowrelease formulations, and mechanical devices. The formulation, route andmethod of administration, and dosage will depend on the disorder to betreated and the medical history of the subject. In general, a dose thatis administered by subcutaneous injection will be greater than thetherapeutically-equivalent dose given intravenously or intramuscularly.Preferably, the dose of compounds of the present invention will beadministered at doses from about 0.1 mg to about 250 mg of body weight.In some embodiments, the dose of compounds of the present invention willbe from about 1 mg to about 60 mg.

The methods of the present invention for treating cancer expressing atleast one MIS receptor, for example expressing MISRII are useful fortreatment of proliferation-related diseases or cancer, which isassociated with cells expressing at least one MIS receptor, for exampleMISRII, comprising contacting a tissue in which proliferation isoccurring, or is at risk for occurring, with the compositions of thepresent invention comprising a therapeutically effective amount ofCompound (I) and/or Compound (II) or functional derivatives thereof.

In some embodiments, the subject treated by the methods of the presentinvention in its many embodiments is a human subject, although it is tobe understood that the principles of the invention indicate that theinvention is effective with respect to all mammals. In this context, amammal is understood to include any mammalian species in which treatmentof diseases associated with cancer or a proliferative-related disorderis desirable, particularly agricultural and domestic mammalian species,as well as transgenic animals.

Administration of Pharmaceutical Compositions

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

After formulation with an appropriate pharmaceutically acceptablecarrier in a desired dosage, the pharmaceutical compositions of thisinvention can be administered to a subject. The pharmaceuticalcompositions of this invention can be administered to a subject usingany suitable means. In general, suitable means of administrationinclude, but are not limited to, topical, oral, parenteral (e.g.,intravenous, subcutaneous or intramuscular), rectal, intracisternal,intravaginal, intraperitoneal, ocular, or nasal routes.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

When the compounds of the present invention, for example Compound (I)and/or Compound (II) are administered as pharmaceuticals, to humans andmammals, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient, i.e., at least one Compound I and/or Compound (II)and/or derivative thereof, in combination with a pharmaceuticallyacceptable carrier.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about 0.1mg to about 250 mg per kilogram of body weight per day, more preferablyfrom about 1 mg to about 60 mg per kg per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The pharmaceutical compositions of the invention include a“therapeutically effective amount” or a “prophylactically effectiveamount” of one or more of the compounds of the present invention, orfunctional derivatives thereof. An “effective amount” is the amount asdefined herein in the definition section and refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result, e.g., a diminishment or prevention ofeffects associated with proliferative disease states or conditions, suchas cancer, wherein the cancer expresses a MIS receptor. Atherapeutically effective amount of the pyrazoloanthrone compounds ofthe present invention or functional derivatives thereof may varyaccording to factors such as the disease state, age, sex, and weight ofthe subject, and the ability of the therapeutic compound to elicit adesired response in the subject. A therapeutically effective amount isalso one in which any toxic or detrimental effects of the therapeuticagent are outweighed by the therapeutically beneficial effects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to, or at an earlier stage of disease, theprophylactically effective amount may be less than the therapeuticallyeffective amount. A prophylatically or therapeutically effective amountis also one in which any toxic or detrimental effects of the compoundare outweighed by the beneficial effects.

The term “synergy” or “synergistic” as used herein, refers to theinteraction of two or more agents so that their combined effect isgreater than each of their individual effects at the same dose alone.

Dosage regimens may be adjusted to provide the optimum desired response(e.g. a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigency of the therapeutic situation. It is especiallyadvantageous to formulate parenteral compositions in dosage unit formfor ease of administration and uniformity of dosage. Actual dosagelevels of the active ingredients in the pharmaceutical compositions ofthis invention may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular subject, composition, and mode ofadministration, without being toxic to the patient.

The term “dosage unit” as used herein refers to physically discreteunits suited as unitary dosages for the mammalian subjects to betreated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the compound, for exampleCompound (I) and/or Compound (II) and/or derivative thereof and theparticular therapeutic or prophylactic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

In some embodiments, therapeutically effective amount can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in other subjects. Generally, the therapeuticallyeffective amount is sufficient to reduce or inhibit cell proliferationin a subject suffering from a proliferative disorder, for examplecancer. In some embodiments, the therapeutically effective amount issufficient to eliminate the proliferative cells, for example eliminatethe cancer cells and/or tumor in a subject suffering cancer and/or aproliferative disease.

Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. The doseadministered to a patient is sufficient to effect a beneficialtherapeutic response in the patient over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose is determined by the efficacy of the particular formulation,and the activity, stability or serum half-life of Compound (I) and/orCompound (II) or functional derivatives thereof, and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular subject. Therapeutic compositions comprising one or more ofCompound (I) and/or Compound (II) or functional derivatives thereof areoptionally tested in one or more appropriate in vitro and/or in vivoanimal models of disease, such as models of cancer, to confirm efficacy,tissue metabolism, and to estimate dosages, according to methods wellknown in the art. In particular, dosages can be initially determined byactivity, stability or other suitable measures of treatment vs.non-treatment (e.g., comparison of treated vs. untreated cells or animalmodels), in a relevant assay. Formulations are administered at a ratedetermined by the LD50 of the relevant formulation, and/or observationof any side-effects of Compound (I) and/or Compound (II) or functionalderivatives thereof at various concentrations, e.g., as applied to themass and overall health of the patient. Administration can beaccomplished via single or divided doses.

In vitro models can be used to determine the effective doses of Compound(I) and/or Compound (II) or functional derivatives thereof as apotential cancer treatment. Suitable in vitro models include, but arenot limited to, proliferation assays of cultured tumor cells, growth ofcultured tumor cells in soft agar (see Freshney, (1987) Culture ofAnimal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch18 and Ch 21), tumor systems in nude mice as described in Giovanella etal., I J. Natl. Can. Inst., 52: 921-30 (1974), mobility and invasivepotential of tumor cells in Boyden Chamber assays as described inPilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesisassays such as induction of vascularization of the chick chorioallantoicmembrane or induction of vascular endothelial cell migration asdescribed in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97 (1999) andLi et al., Clin. Exp. Metastasis, 17:423-9 (1999), respectively.Suitable tumor cells lines are available, e.g. from American Type TissueCulture Collection catalogs.

In vivo models are the preferred models to determine the effective dosesof Compound (I) and/or Compound (II) or functional derivatives thereofas disclosed herein as potential cancer treatments. Suitable in vivomodels include, but are not limited to, mice that carry a mutation inthe KRAS oncogene (Lox-Stop-Lox K-RasGi2D mutants, Kras24TYj) availablefrom the National Cancer Institute (NCI) Frederick Mouse Repository.Other mouse models known in the art and that are available include butare not limited to models for breast cancer, gastrointestinal cancer,hematopoietic cancer, lung cancer, mammary gland cancer, nervous systemcancer, ovarian cancer, prostate cancer, skin cancer, cervical cancer,oral cancer, and sarcoma cancer (seehttp://emice.nci.nih.gov/mouse_models/).

In determining the effective amount of Compound (I) and/or Compound (II)or functional derivatives thereof to be administered in the treatment orprophylaxis of disease the physician evaluates circulating plasmalevels, formulation toxicities, and progression of the disease.

The efficacy and toxicity of the compound can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose is effective in 50% of the population) and LD50(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index, and it can be expressedas the ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. In some embodiments, the compounds ofthe present invention have an ED₅₀ value ranging from 0.01-10 μM in anassay for JNK. The compound (I) as a IC₅₀ of about 0.11 μM for JNK1 andJNK2, and about 0.15 μM for JNK3 according to the assay as disclosed inU.S. Pat. No. 7,119,114, which is incorporated herein in its entirety byreference.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of the compounds of the presentinvention, for example Compound (I) and/or Compound (II) and/orfunctional derivatives thereof of the invention is 0.1-250 mg/kg, and insome embodiments, the dosage is 1-60 mg/kg. It is to be noted thatdosage values may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

The invention features an article of manufacture that contains packagingmaterial and compounds of the present invention, for example Compound(I) and/or Compound (II) and/or functional derivatives thereof in aformulation contained within the packaging material. This formulationcontains an at least one of the compounds of the present invention, forexample Compound (I) and/or Compound (II) and/or functional derivativesthereof and the packaging material contains a label or package insertindicating that the formulation can be administered to the subject totreat one or more conditions as described herein, in an amount, at afrequency, and for a duration effective to treat or prevent suchcondition(s). Such conditions are mentioned throughout the specificationand are incorporated herein by reference. Suitable compounds of thepresent invention, for example Compound (I) and/or Compound (II) and/orfunctional derivatives thereof are described herein.

More specifically, the invention features an article of manufacture thatcontains packaging material and at least one of the compounds of thepresent invention, for example Compound (I) or functional derivativesthereof contained within the packaging material. The packaging materialcontains a label or package insert indicating that the formulation canbe administered to the subject to alleviate a proliferative disorder,for example cancer in an amount, at a frequency, and for a durationeffective treat or prevent symptoms associated with such disease statesor conditions discussed throughout this specification.

Pharmaceutical Compositions

In another embodiment of the invention, pharmaceutical compositionscontaining one or more compounds of this invention are disclosed. Forpurpose of administration, a compound of structure (I) and/or structure(II) is preferably formulated as a pharmaceutical composition.Pharmaceutical compositions of the present invention comprise a compoundof this invention and a pharmaceutically acceptable carrier, wherein thecompound is present in the composition in an amount which is effectiveto treat the condition of interest. Preferably, the pharmaceuticalcompositions of the present invention include a compound of structure(I) and/or structure (II) in an amount from 0.1 mg to 250 mg per dosagedepending upon the route of administration, and more typically from 1 mgto 60 mg. Appropriate concentrations and dosages can be readilydetermined by one skilled in the art.

Pharmaceutically acceptable carriers are familiar to those skilled inthe art. For compositions formulated as liquid solutions, acceptablecarriers include saline and sterile water, and may optionally includeantioxidants, buffers, bacteriostats and other common additives. Thecompositions can also be formulated as pills, capsules, granules, ortablets which contain, in addition to a compound of this invention,diluents, dispersing and surface active agents, binders, and lubricants.One skilled in this art may further formulate the compounds of thisinvention in an appropriate manner, and in accordance with acceptedpractices, such as those disclosed in Remington's PharmaceuticalSciences, Gennaro, Ed., Mack Publishing Co., Easton, Pa. 1990.

While it is possible for compounds of the present invention, for exampleCompound (I) and/or Compound (II) and/or functional derivatives thereof,to be administered alone, it is preferable to administer the compound asa pharmaceutical composition.

Formulations of the invention can be prepared by a number or means knownto persons skilled in the art. In some embodiments the formulations canbe prepared by combining (i) at least Compound (I) and/or and/orCompound (II) and/or functional derivatives thereof in an amountsufficient to provide a plurality of therapeutically effective doses;(ii) the water addition in an amount effective to stabilize each of theformulations; (iii) the propellant in an amount sufficient to propel aplurality of doses from an aerosol canister; and (iv) any furtheroptional components e.g. ethanol as a cosolvent; and dispersing thecomponents. The components can be dispersed using a conventional mixeror homogenizer, by shaking, or by ultrasonic energy. Bulk formulationcan be transferred to smaller individual aerosol vials by using valve tovalve transfer methods, pressure filling or by using conventionalcold-fill methods. It is not required that a stabilizer used in asuspension aerosol formulation be soluble in the propellant. Those thatare not sufficiently soluble can be coated onto the drug particles in anappropriate amount and the coated particles can then be incorporated ina formulation as described above.

The compositions of the present invention can be in any form. Theseforms include, but are not limited to, solutions, suspensions,dispersions, ointments (including oral ointments), creams, pastes, gels,powders (including tooth powders), toothpastes, lozenges, salve, chewinggum, mouth sprays, pastilles, sachets, mouthwashes, aerosols, tablets,capsules, transdermal patches, that comprise one or more of thecompounds of the present invention, and/or their functional derivativesthereof for oral or subcutaneous administration.

In certain embodiments, the compounds of the present invention, forexample Compound (I) and functional derivatives thereof are administeredto a subject as a pharmaceutical composition with a pharmaceuticallyacceptable carrier. In certain embodiments, these pharmaceuticalcompositions optionally further comprise one or more additionaltherapeutic agents. In certain embodiments, the additional therapeuticagent or agents are anti-cancer agents. In some embodiments, thetherapeutic agents are chemotherapeutic agents, for example but notlimited to, cisplatin, paxicital etc. In some embodiments, thetherapeutic agents are radiotherapeutic agents. Examples ofchemotherapeutic agents in the pharmaceutical compositions of thisinvention are, for example nitrogen mustards such as cyclophosphamide,ifosfamide, and melphalan; ethylenimines and methylmelamines such ashexamethylmelamine and thiotepa; pyrimidine analogs such as fluorouraciland fluorodeoxyuridine; vinca alkaloids such as vinblastine;epipodophyllotoxins such as etoposide and teniposide; antibiotics suchas actinomycin D, doxorubicin, bleomycin, and mithramycin; biologicalresponse modifiers such as interferon, platinum coordination complexessuch as cisplatin and carboplatin; estrogens such as diethylstilbestroland ethinyl estradiol; antiandrogens such as flutamine; and gonadotropinreleasing hormone analogs such as leuprolide. Other compounds such asdecarbazine, nitrosoureas, methotrexate, diticene, and procarbazine arealso effective and encompassed for use in the methods of the presentinvention. Of course, other chemotherapeutic agents which are known tothose of ordinary skill in the art can readily be substituted as thislist should not be considered exhaustive or limiting.

In some embodiments the pharmaceutical composition comprises compoundsof the present invention, for example Compound (I) and/or Compound (II)and/or functional derivatives thereof, alone or in any plurality ofcombinations. In other embodiments, the pharmaceutical compositionsoptionally further comprise one or more additional therapeutic agentsincluding but not limited to paxicital, functional derivatives ofpaxicital, cisplatin and functional derivatives of cisplatin, andinterferons.

In some embodiments, the pharmaceutical composition comprising Compound(I) and/or Compound (II) or derivatives thereof as disclosed herein cansupplement the treatment of any known additional therapy, including, butnot limited to, antibody administration, vaccine administration,administration of cytotoxic agents, natural amino acid polypeptides,nucleic acids, nucleotide analogues, and biologic response modifiers. Insome embodiments, additional therapy is, for example, surgery,chemotherapy, radiotherapy, thermotherapy, immunotherapy, hormonetherapy and laser therapy. In some embodiments, the additional therapyis chemotherapy. Two or more combined compounds may be used together orsequentially with the pharmaceutical composition comprising Compound (I)and/or Compound (II) or derivatives thereof. In some embodiments,Compound (I) and/or Compound (II) or derivatives thereof can beadministered before the additional therapy, after the additional therapyor at the same time as the additional therapy. In some embodiments, theCompound (I) and/or Compound (II) or functional derivatives thereof areadministered a plurality of times, and in other embodiments, theadditional therapies are also administered a plurality of times.

In some embodiments, Compound (I) and/or Compound (II) or functionalderivatives thereof can also be administered in therapeuticallyeffective amounts as a portion of an anti-cancer cocktail. Ananti-cancer cocktail is a mixture, for example at least one of compound(I) and/or Compound (II) or functional derivatives thereof is combinedwith one or more additional anti-cancer agents in addition to apharmaceutically acceptable carrier for delivery. The use of anti-cancercocktails as a cancer treatment is routine. Anti-cancer agents that arewell known in the art and can be used as a treatment in combination withCompound (I) and/or Compound (II) or functional derivatives thereof asdisclosed herein include, but are not limited to: Actinomycin D,Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin,Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide,Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin,Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium,Etoposide (V16-213), Flosuridine, S-Fluorouracil (5-Fu), Flutamide,Hydroxyurea (hydroxycarb amide), Ifosfamide, Interferon Alpha-2 a,Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog),Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan,Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl,Ockeotide, Paclitaxel; Plicamycin, Procarbazine HCl, Streptozocin,Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate,Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine,Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, andVindesine sulfate, and analogues thereof.

In certain embodiments, the pharmaceutical compositions comprisingcompound (I) and/or compound (II) or functional derivatives thereof canoptionally further comprise one or more additional therapies or agents.In certain embodiments, the additional agent or agents are anti-canceragents. In some embodiments, the therapeutic agents are chemotherapeuticagents, for example cisplatin, paxicital etc. In some embodiments, thetherapeutic agents are radiotherapeutic agents. Examples ofchemotherapeutic agents in the pharmaceutical compositions of thisinvention are, for example nitrogen mustards such as cyclophosphamide,ifosfamide, and melphalan; ethylenimines and methylmelamines such ashexamethylmelamine and thiotepa; pyrimidine analogs such as fluorouraciland fluorodeoxyuridine; vinca alkaloids such as vinblastine;epipodophyllotoxins such as etoposide and teniposide; antibiotics suchas actinomycin D, doxorubicin, bleomycin, and mithramycin; biologicalresponse modifiers such as interferon, platinum coordination complexessuch as cisplatin and carboplatin; estrogens such as diethylstilbestroland ethinyl estradiol; antiandrogens such as flutamine; and gonadotropinreleasing hormone analogs such as leuprolide. Other compounds such asdecarbazine, nitrosoureas, methotrexate, diticene, and procarbazine arealso effective. Of course, other chemotherapeutic agents which are knownto those of ordinary skill in the art can readily be substituted as thislist should not be considered exhaustive or limiting.

In some embodiments, compound (I) and/or Compound (II) or functionalderivatives thereof is administered to a subject with other anti-cancertherapies, for example cancer therapies to which the cancer waspreviously resistant or refractory.

In some embodiments, the methods of the present invention are directedto use of pyrazoloathrone and functional derivatives thereof with othertherapeutic agents, for example chemotherapy agents, wherein thechemotherapy agents, for example paclitaxel or MIS can be used at alower dose that results in decreased side effects.

In some embodiments, the chemotherapeutic agent is MIS, for examplerecombinant human MIS (rhMIS). In such embodiments, MIS or rhMIS can beprepared and administered, in any form, by any method known by personsof ordinary skill in the art, for example as disclosed in InternationalPatent Application WO92/18152 and European Patent EP584287 and alsodisclosed in patent Applications WO94/00133 and EP221761, which areincorporated herein in their entity by reference.

In certain embodiments, the endogenous compounds are isolated and/orpurified or substantially purified by one or more purification methodsdescribed herein or known by those skilled in the art. Generally, thepurities are at least 90%, in particular 95% and often greater than 99%.In certain embodiments, the naturally occurring compound is excludedfrom the general description of the broader genus.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can perform itsintended function. The term “pharmaceutically acceptable carriers” isintended to include all solvents, diluents, or other liquid vehicle,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Typically, such compounds are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: sugars, such as lactose, glucose andsucrose; starches, such as corn starch and potato starch; cellulose, andits functional derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

In certain embodiments, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts, esters,amides, and prodrugs as used herein refers to those carboxylate salts,amino acid addition salts, esters, amides, and prodrugs of the compoundsof the present invention which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use of the compounds of the invention. The term “salts”refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention.

These salts can be prepared in situ during the final isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. These may include cations based onthe alkali and alkaline earth metals, such as sodium, lithium,potassium, calcium, magnesium and the like, as well as non-toxicammonium, quaternary ammonium, and amine cations including, but notlimited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. (See, for example, Berge S. M., et al., “PharmaceuticalSalts,” J. Pharm. Sci., 1977; 66:1-19 which is incorporated herein byreference).

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters.

As used herein, “pharmaceutically acceptable salts or prodrugs are saltsor prodrugs that are, within the scope of sound medical judgment,suitable for use in contact with the tissues of subject without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use.These compounds include the zwitterionic forms, where possible, ofcompounds of the invention.

The term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the present invention. Thesesalts can be prepared in situ during the final isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. These may include cations based onthe alkali and alkaline earth metals, such as sodium, lithium,potassium, calcium, magnesium and the like, as well as non-toxicammonium, quaternary ammonium, and amine cations including, but notlimited to ammonium, tetramethylammonium, tetraethyl ammonium, methylamine, dimethyl amine, trimethylamine, triethylamine, ethylamine, andthe like (see, e.g., Berge S. M., et al. (1977) J. Pharm. Sci. 66, 1,which is incorporated herein by reference).

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the compounds of the invention, for example thepyrazoloathrone and functional derivatives thereof of the invention, byhydrolysis in blood. A thorough discussion is provided in T. Higachi andV. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in: Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987, both of which are hereby incorporated by reference. As usedherein, a prodrug is a compound that, upon in vivo administration, ismetabolized or otherwise converted to the biologically, pharmaceuticallyor therapeutically active form of the compound. The prodrug may bedesigned to alter the metabolic stability or the transportcharacteristics of a compound, to mask side effects or toxicity, toimprove the flavor of a compound or to alter other characteristics orproperties of a compound. By virtue of knowledge of pharmacodynamicprocesses and drug metabolism in vivo, once a pharmaceutically activecompound is identified, those of skill in the pharmaceutical artgenerally can design prodrugs of the compound (see, e.g., Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press,N.Y., pages 388-392). Conventional procedures for the selection andpreparation of suitable prodrugs are described, for example, in “Designof Prodrugs,” ed. H. Bundgaard, Elsevier, 1985. Suitable examples ofprodrugs include methyl, ethyl and glycerol esters of the correspondingacid.

In other embodiments of the present invention, the pyrazoloathrone andfunctional derivatives thereof are conjugated or covalently attached toanother targeting agent to increase the specificity of a pyrazoloathroneand functional derivatives thereof targeting the cell, for example acancer cell. Targeting agents can include, for example withoutlimitation, antibodies, cytokines and receptor ligands. In someembodiments, the targeting agent is overexpressed on the cells to betargeted, for example the cancer cells as compared to normal cells. Inalternative embodiments, the pyrazoloathrone and functional derivativesthereof can be conjugated or covalently attached to compounds thatelicit an immune response, such as for example but without limitation,cytokines.

In some embodiments, the pyrazoloathrone and functional derivativesthereof of the present invention can be conjugated to, by covalentlinkage or any other means, to another agent, for example a chemotherapyagent, or MIS or functional derivatives and analogues thereof. In someembodiments, the pyrazoloathrone and functional derivatives thereof ofthe present invention can be conjugated to a targeting moiety, forexample a cancer cell targeting moiety to target the compounds of thepresent invention to a cancer cell. Such targeting moieties and methodsare well known by persons of ordinary skill in the art and areencompassed for use in the methods of the present invention. Theconjugation may be a permanent or reversible conjugation.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients. Inone aspect, a solution of resolvin and/or protectin or precursor oranalog thereof can be administered as eye drops for ocularneovascularization or ear drops to treat otitis.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs.

In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

In some instances, pharmaceutical compositions comprising the resolvinsand protectins of the invention for the administration of angiogenesismay be in a formulation suitable for rectal or vaginal administration,for example as a suppository, which may be prepared by mixing one ormore compounds of the invention with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and, thereforerelease the active compound. Suitable carriers and formulations for suchadministration are known in the art.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof. Powders and sprays cancontain, in addition to a compound of this invention, excipients such aslactose, talc, silicic acid, aluminum hydroxide, calcium silicates andpolyamide powder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of the compounds (resolvins and/or protectins and/or precursorsor analogues thereof) of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art.

More specifically, the invention features an article of manufacture thatcontains packaging material and at least one compound of the presentinvention, for example Compound (I) and/or Compound (II) and/orfunctional derivatives thereof are contained within the packagingmaterial. The packaging material contains a label or package insertindicating that the formulation can be administered to the subject withneovascularization in an amount, at a frequency, and for a durationeffective treat or prevent symptoms associated with such disease statesor conditions discussed throughout this specification. In someembodiments, the proliferative disorder is a cancer. In someembodiments, the cancer is for example a MIS-responsive cancer and/orexpresses MISRII, for example ovarian cancer.

Remington's Pharmaceutical sciences Ed. Germany, Merk Publishing,Easton, Pa., 1995 (the contents of which are hereby incorporated byreference), discloses various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its functional derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; malt;gelatin; talc; excipients such as cocoa butter and: suppository waxes;oils such as peanut oil, cottonseed oil; safflower oil; sesame oil;olive oil; corn oil and soybean oil; glycols; such a propylene glycol;esters such as ethyl oleate and ethyl laurate; agar; buffering agentssuch as magnesium hydroxide and aluminum hydroxide; water; isotonicsaline; Ringer's solution, ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium sulfate, as well as coloring agents,releasing agents, coating agents, sweetening, flavoring and perfumingagents, preservatives and antioxidants can also be present in thecomposition, according to the judgment of the formulator.

Uses

In another embodiment, the present invention provides a method fortreating a variety of conditions by administering an effective amount ofa pyrazoloathrone and functional derivatives thereof of the invention toa subject in need thereof. Conditions that may be treated by thecompounds of this invention, or a pharmaceutical composition containingthe same, include any condition which is treated or reduces the symptomsby administration of MIS or activation of MIS signaling or activation ofMISRII, and thereby benefit from administration of a pyrazoloathrone andfunctional derivatives thereof. Representative conditions in this regardinclude, for example, but not limited to, cancers that express MISreceptors, for example cancer that express MISRII, for example, but notlimited to ovarian, cervical and endometrial cancer. Other conditionswhich can be treated with MIS or activation of MIS signalling reducesthe symptoms are, for example, rheumatoid arthritis, proliferativediseases such as cancer, treatment of prostatic cancer, polycysicovarian disease, benign prostatic hypertrophy and precocious puberty andother hyperandrogen disorders such as testitoxicosis.

Accordingly, the present invention relates to the use of pyrazoloathroneand functional derivatives thereof for the treatment of any disorderwhere administration of the MIS protein or a nucleic acid encoding MISprotein or a functional derivative of MIS or activation MISRII is whole,or part, of the therapeutic regime. In some embodiments, thepyrazoloathrone is SP600125 (or compound 1 herein), as disclosed in U.S.Pat. No. 7,119,114 which is incorporated in its entirety herein byreference. In other embodiments, the pyrazoloathrone is a derivative ofpyrazoloathrone, for example substituted antra(1,9-cd)pyrazol-6(2H)-one,and anthrapyrazolone derivatives as disclosed in European Patent EP0103381 and EP0700390, which are incorporated in their entirety hereinby reference.

In some embodiments, the methods of the present invention are directedto use of pyrazoloathrone and functional derivatives thereof with othertherapeutic agents, for example chemotherapy agents, wherein thechemotherapy agents, for example paclitaxel or MIS can be used at alower dose that results in decreased side effects.

Uses of Pyrazoloanthrone or Functional Derivatives or Analogues Thereoffor the Treatment of Excess Androgen States

Another embodiment, the pyrazoloanthrone or functional derivatives oranalogues thereof, such as SP600125 can be used for the treatment of adisorder associated with excess androgen production in a subject. Theinventors have previously demonstrated that the administration of MISprotein and/or MIS nucleic acid decreases levels of androgen in asubject, and decreases serum levels of androgen in a subject, asdisclosed in U.S. Pat. No. 6,673,352 and U.S. patent application Ser.No. 10/683,346, which are incorporated herein in their entirety byreference. Transgenic mice that overexpress MIS have also shown to havedecreased serum testosterone concentrations, and administration of MISresults in decreased serum testosterone levels (Sriraman et al., J.Androl. 2001, 22(5):750-8 and Trbovich et al., PNAS, 2001 Mar. 13;98(6):3393-7). MIS has also been demonstrated to suppress bothandrogen-stimulated growth and androgen-independent survival of cells,and MIS regulates prostate growth by suppressing testicular testosteronesynthesis also direct regulates androgen-induced gene expression andgrowth in the prostate at the cellular level (Trann et al, Mol.Endocrinol. 2006, 20(10):2382-91). Accordingly, in another embodiment,the present invention also relates to methods to use the

Androgen stimulates or controls the development and maintenance ofmasculine characteristics in vertebrates by binding to androgenreceptors. Androgens are also known as androgenic hormones or testoids,and are also the precursor of all estrogens, the female sex hormones.The primary and most well-known androgen is testosterone.

Without wishing to be bound by theory, excessive androgen production bythe adrenal glands and/or the ovary, results in androgen excess and canresult from increased local tissue sensitivity to circulating androgens.Androgen excess affects different tissues and organ systems, causingclinical conditions ranging from acne to hirsutism to frankvirilization.

Hyperandrogenism, which refers to the excess production and secretion ofandrogens and precursors, is a common and sometimes seriousendrocrinopathy for women of reproductive age. The excess androgens andprecursors originate from the adrenal glands and ovaries in variousproportions and manifest in varying effects depending on the amount ofexcess androgen. Clinical manifestations range from hirsutism (excessivehair growth of male pattern, sometimes accompanied by acne) tovirilization (clitorimegaly, temporal balding, deepening of voice, orenhanced musculature).

Hyperandrogenism occurs as part of a wide spectrum of diseasemanifestations, including polycystic ovary syndrome (PCOS) which is avariable combination of hirsutism, infertility, obesity, insulinresistance and polycystic ovaries, the HAIR-AN syndrome(hyperandrogenism, insulin resistance and acanthosis nigricans), ovarianhyperthecosis (HAIR-AN with hyperplasia of luteinized theca cells inovarian stroma), and other manifestations of high intraovarian androgenconcentrations (e.g., follicular maturation arrest, atresia,anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility),androgenproducing tumors (virilizing ovarian or adrenal tumors).

Hirsutism is excessive recognizable hair growth characterized by anincrease in the number and length of terminal hairs inandrogen-sensitive areas. Racial, familial, genetic, and ethnicdifferences all affect the occurrence of hirsutism. Hirsutism isdifficult to quantitate. The entire body needs to be inspected and thefindings must be documented carefully. Particular attention should bedirected to the chin, lip, sideburns, breasts, and sternum, the midlinebetween the umbilicus and the pubis and the thigh.

Ferriman and Gallwey published a rating scale for grading hirsutism andis commonly known by persons of ordinary skill in the art. This scaleallows the physician to measure a response to therapy objectively. Thissystem is the most widely used and evaluates body areas forabsent-to-severe hirsutism with scores of 0-4, respectively. Scores of 8and higher are consistent with a diagnosis of hirsutism. This scale doesnot measure the thickness of the hair, which is another way ofobjectively assessing excess hair. Scoring systems are a useful aid inquantifying hirsutism and in evaluating treatment response. Even withscores greater than 8, the patient provides the definition. From aclinical standpoint, the patient can determine if he or she notices adifference. Photographs are helpful for documentation and for followingthe progress of therapy.

Virilization is relatively uncommon; it occurs with extremehyperandrogenism. Virilization is characterized by temporal balding,breast atrophy, androgenic muscle development, clitoral hypertrophy,amenorrhea, deepening of the voice, and extreme hirsutism.

Current medical therapies for women are directed against the adrenals,the ovaries or the androgen receptor. Glucocorticoid therapy is directedagainst the adrenal glands but is limited, in some cases, by unwantedsuppression of cortisol synthesis. GnRH therapy is directed against theovaries, but is expensive, and its long-term effects are unknown.Further, therapy using oral contraceptives may be unsuitable becausemost contain progestins with androgenic activity.

Because the abnormal production of androgens is implicated in thepathways of many diseases and/or disorders for which there are noacceptable treatments, a need exists to find small molecules to inhibitthe production of gonadotropins and/or androgens in mammals for theirtreatment and/or prophylaxis.

Accordingly, in one embodiment, the pyrazoloanthrone or functionalderivatives or analogues thereof, such as SP600125 can be used for thetreatment of a disorder associated with excess androgen production in asubject.

The term “androgen” is used herein to mean steroids that encourage thedevelopment of male sex characteristics and include the steroidderivatives of androstane including, testosterone, androstenedione, andanalogs.

As used herein, a disease state or disorder characterized by “androgenicdependency” is a disease state which is exacerbated by, or caused by,insufficient, excessive, inappropriate or unregulated androgenproduction. Examples of such diseases in men include, but are notlimited to, BPH, metastatic prostatic carcinoma, testicular cancer,androgen dependent acne, male pattern baldness and precocious puberty inboys. Examples of such diseases in women include, but are not limitedto, hyperandrogenism, hirsutism, virilization, POCS, HAIR-AN syndrome,ovarian hyperthecosis, follicular maturation arrest, atresia,anovulation, dysmenorrhea, dysfunctional uterine bleeding, infertility,androgen-producing tumors.

As used herein, “androgen inhibiting” refers to an effective amount ofan the pyrazoloanthrone or functional derivatives or analogues thereofas defined herein, such as SP600125, which will cause a decrease in thein vivo levels of the androgen to normal or sub-normal levels, whenadministered to a subject for the prophylaxis or treatment of a diseasestate which is exacerbated by, or caused by, excessive or unregulatedandrogen production.

In some embodiments, the pyrazoloanthrone or functional derivatives oranalogues thereof as disclosed herein, such as SP600125, can be used totreat prostate cancer. The impact of androgens on prostate carcinoma isknown, as is the treatment of prostate cancer by androgen deprivation,including androgen blockade and inhibition of androgen synthesis(Huggins et al., Archs. Surg., Vol. 43, pp. 209-223 (1941)). J. SteroidBiochem. Molec. Biol., Vol. 37, pp. 349-362 (1990)). In addition,steroid hormones are widely used as contraceptives.

Anti-spermatogenic agents are male contraceptives that inhibitspermatogenesis, the process leading to mature spermatazoa. Drugs thatinterfere in this process include androgens and anti-androgens. Sincethe anti-androgenic effects of the pyrazoloanthrone or functionalderivatives or analogues thereof as disclosed herein, such as SP600125,are reversible, the compounds can also be used as a male contraceptiveagent. Korolkovas, A., Essentials Of Medicinal Chemistry, SecondEdition, pp. 1032 (1988).

In some embodiments, other agents can be used in combination with thepharmaceutical compositions comprising pyrazoloanthrone or functionalderivatives or analogues thereof as disclosed herein for the treatmentof excess androgen in a subject. In some embodiments, the agentsfunction to lower the serum-free androgen levels and blocking theperipheral androgen action. Examples of such agents include, but are notlimited to, suppression of ovarian androgens by administration ofestrogens and/or progestins (i.e., contraceptive pill) or GnRH agonistand add-back estrogen therapy; suppression of adrenal androgens byadministration of glucocorticoids (such as dexamethasone, prednisolone),antiandrogens (such as spironolactone, flutamide, cyproterone acetate),5α-reductase inhibitor (such as finasteride), bromocriptine, andinsulin-sensitizing drugs (such as metformin, thiazolidinediones).

Subjects amenable to treatment of the pyrazoloanthrone or functionalderivatives or analogues thereof by the methods as disclosed herein aresubjects that have been identified with a disease or disorder associatedwith excess androgen levels, such as, for example disorders such as, butnot limited to BPH, prostate carcinoma, benign prostic hypertrophy,testicular cancer, androgen dependent acne, male pattern baldness,precocious puberty, hyperandrogenism, hirsutism, virilization, POCS,HIAR-AN syndrome, ovarian hyperthecosis, follicular maturation arrest,atresia, anovulation, dysmenorrheal, dysfunctional uterine bleeding,infertility and androgen-producing tumors.

In some embodiments, subjects amenable to treatment of thepyrazoloanthrone or functional derivatives or analogues thereof by themethods as disclosed herein are subjects with congenical adrenalhyperplasma (CAH), which can be commonly identified by one of ordionaryskill in the art. CAH is most typically an autosomal recessive disorderwhere the enzyme 21-hydrolase is missing or functionally deficent.Alternatively subjects with CAH can have a loss and/or reduction in thefunction of 11α-hydroxylase enzyme and/or a 3α-hydroxy-steroiddehydrogenase enzyme. When these enzymes are missing or functioning atlow levels, the body cannot make adequate amounts of the adrenal steroidhormones cortisol and aldosterone. High levels of ACTH that stimulateadrenal hyperplasia and hypersecretion of androgen precursors forcortisol and aldosterone synthesis ensue. CAH can appear in utero ordevelop postnatally. Pseudohermaphroditism may be present at birth.

The 21-hydroxylase deficiency is the most common autosomal-recessivedisorder (more common than cystic fibrosis) and manifests itself withelevated levels of 17-hydroxyprogesterone. The 11a-hydroxylasedeficiency is characterized by elevated levels of 11-deoxy-cortisol(compound S) and results in elevated levels of deoxycorticosterone(DOC), a mineralocorticoid. Hypertension and hypokalemia can be aprominent feature of 11a-hydroxylase deficiency. Another form of CAH,3a-hydroxy-steroid dehydrogenase deficiency, results in elevated levelsof pregnenolone, 17-hydroxy-pregnenolone, and DHEA. This condition islethal if not detected because no corticosteroids are synthesized.

A partial defect in the above enzymes that manifests after pubertyresults in elevated levels of adrenal steroids via the same mechanism.The elevations are not as marked as they are with the congenitalcondition and this condition is referred to as nonclassical(maturity-onset or late-onset) CAH. Accordingly, in some embodiments,subjects amenable to treatment of the pyrazoloanthrone or functionalderivatives or analogues thereof by the methods as disclosed herein aresubjects with nonclassical (maturity-onset or late onset) CAH.

In some embodiments, subjects amenable to treatment of thepyrazoloanthrone or functional derivatives or analogues thereof by themethods as disclosed herein are female subjects with testosterone levelsabout or exceeding 2.0 ng/mL (200 ng/dL, 8.92 nmol/L) or at least about2.5 times the upper limit of the reference range. In some embodiments,such subjects have Sertoli-Leydig cell tumors, hilus cell tumors, andlipoid cell (adrenal rest) tumors are the most common. Sertoli-Leydigcell tumors reach palpable size at the time of clinical diagnosis,whereas hilar cell and lipoid cell tumors are difficult to detect by anymeans because of their small size.

In some embodiments, subjects amenable to treatment of thepyrazoloanthrone or functional derivatives or analogues thereof by themethods as disclosed herein are subjects with tumors of the adrenalglands (adenomas, carcinomas), which secrete elevated levels ofandrogens. In such embodiments, such subjects amenable to treatment bythe methods as disclosed herein can be identified by having a DHEASlevel of about or exceeding 7 μg/mL (18 μmol/L).

Other subjects that are amenable to the methods of treatment of excessandrogen states as disclosed herein include, for example, classical andnonclassical (late-onset) CAH, cushing syndrome, where subjects withCushing syndrome secrete elevated androgens, Hyperandrogenic, insulinresistance, and acanthosis nigricans (HAIR-AN) syndrome. In someembodiments, other subjects amenable to the methods of treatment ofexcess androgen states as disclosed herein include, for example,subjects with mild androgenic disorders, such as, but not limited to,Ovulatory PCOS (Ovulatory hyperandrogenic subjects with polycystic ovaryat ultrasonography), Idiopathic hyperandrogenism (an oOvulatoryhyperandrogenic subject but with normal ovaries at ultrasonography);Idiopathic hirsutism (subjects with an androgenic phenotype with normalandrogens).

Reference testosterone levels and DHEAS levels are commonly known bypersons of ordinary skill in the art, and are disclosed in Guay et al,International Journal of Impotence Research (2004) 16, 112-120, which isincorporated herein in its entirety by reference. Briefly, normalandrogen levels in women between the ages of 20 and 49 years rangebetween; DHEAS; about 195.6-140.4 ug/dl; serum testosterone about51.5-33.7 ng/dl and free testosterone 1.51-1.03 pg/ml. Accordingly,subjects amenable to the treatment of the pyrazoloanthrone or functionalderivatives or analogues thereof by the methods as disclosed herein haveat least about a 20%, or at least about a 30% or at least about a 40% orat least about a 50%, or at least about a 60% or at least about a 70%,or at least about a 80%, or at least about a 90%, or at least about a100% or greater increase in DHEAS or serum testosterone, or freetestosterone levels as compared to the highest range value of the normalvalue for DHEAS (195.6 μg/dl), serum testosterone (51.5 ng/dl), freetestostereone (1.51 pg/ml). In some embodiments, subjects amenable tothe treatment of the pyrazoloanthrone or functional derivatives oranalogues thereof by the methods as disclosed herein have at least abouta 2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold, or at least about a 10-fold or greater increase inDHEAS or serum testosterone, or free testosterone levels as compared tothe highest range value of the normal value for DHEAS (195.6 μg/dl),serum testosterone (51.5 ng/dl), free testostereone (1.51 pg/ml).

DHEAS can be measured by one of ordinary skill in the art using a kitfrom by Diagnostic Products Corporation of Los Angeles, Calif., USA.Cross-reactivity has previously been determined as being 100% for DHEASand 0.121% with androstenedione, 15% with 9-hydroxyandrostenedione,0.046% with estrone 3 sulfate, 0.55% with androsterone sulfate, 0.5%with DHEA and negligible for all other steroids tested. FreeTestosterone can be measured by one of ordinary skill in the art usingwas measured using the Coat a Count Kits of Diagnostic ProductsCorporation, Los Angeles, Calif., USA. Cross-reactivity has previouslybeen determined to be 0.41% for dihydrotestosterone, 0.01% forandrostenedione, 0.10% for methyl testosterone ando0.01% for all othersteroids tested. Total serum testosterone levels can be measured by oneof ordinary skill in the art using with the Immunochem serumtestosterone kit of ICN Biomedicals Inc., Diagnostic Division of CostaMesa, Calif., USA.

The assays to determine serum pregnenolone and 17-hydroxypregnenolonecan be performed by one of ordinary skill in the art from the kit fromQuest Laboratory in Tarzana, Calif., USA. Free Androgen Index (FAI) canbe calculated using the following formula:

(Total testosterone ng/dl×0.0347)/(SHBG nmol/l)×100=FAI

Assay for Identifying MIS Mimetics

In another aspect, the present invention is the first high throughputquantitative assay directed to methods of screening for agents ormolecules that function as a mimetic of MIS or activate the MISRII oractivate MISRII-mediated signaling. In some embodiments, the presentinvention provides methods an assay, wherein the agent is contacted witha cell comprising a nucleic acid construct comprising a BMP-responsiveelement operatively linked to a nucleic acid encoding a reporter gene.In some embodiments, the reporter gene encodes a protein withfluorescent activity and/or chromogenic activity, for example but notlimited to fluorescent proteins, for example green fluorescent protein(GFP) or variants thereof or bioluminescent proteins, for exampleluciferase or variants thereof. In some embodiments, the cell alsocomprises a nucleic acid construct comprising MISRII. In someembodiments, the agent is a small molecule, nucleic acid, nucleic acidanalogue, aptamer, ribosome, peptide, protein, antibody or variants orfragments thereof. An agent that functions as a MIS mimetic orfunctional derivative of MIS or activates MISRII-mediated signaling willresult in a change in the signal from the reporter gene, where thechange is a result of contacting the cell with agent compared to whenthe cell is not contacted with the agent.

In some embodiments, the method involves the introduction into a cell ofa nucleic acid construct encoding a reporter gene which is operativelylinked to a BMP-responsive element (BME) or a fragment thereof. TheBMP-responsive element on the mouse (M. mus) genome is located onchromosome 2 (Chr2) and corresponds to RefSeq No: NT_(—)039207.693504644-93506272 (SEQ ID NO:1). Accordingly, the reporter gene can beoperatively linked to a nucleic acid encoding a SEQ ID:1, or a fragmentor derivative thereof. In some embodiments, the BMP response elements isa homologue or cognate of SEQ ID NO:1, for example but not limited to ahuman or other primate homologue of SEQ ID NO:1. The introduction of thenucleic acid can be by any method described commonly known by personsskilled in the art, for example vectors, viral vectors, transfection,naked DNA etc. In some embodiments, the method also involvesintroduction into the cells of a nucleic acid construct encoding MISRIIor a homologue or fragment thereof, operatively linked to a regulatorysequence. In some embodiments, the regulatory sequence is a promoter,and in some embodiments the promoter is constitutively expressed and inalternative embodiments the promoter is an inducible promoter, a tissuespecific promoter and/or a developmentally regulated promoter.

Cells from any species and any tissue can be used to carry out themethods of the invention. The cells are cultured or maintained in aconventional culture medium under suitable conditions permitting growthof the cells. For example, the cell is cultured in standard tissueculture media containing the necessary reagents to select for cellswhich stably retain the nucleic acid construct described above. Cellsmay be cultured in standard tissue culture dishes e.g. multidishes andmicrowell plates, or in other vessels, as desired. In someconfigurations, the assay can be conducted in a 96 well; 386-well orother multi-well plates.

In one embodiment, agents are added to the culture media to assess theireffect on the signal produced from the reporter gene within the cell.Cells at this time may be bathed in tissue culture media (with orwithout serum) or balanced salt solution. The agent may be applied tothe media, wherein it contacts the cell and induces its effects or itmay be intracellular within the cell. In one embodiment, the agent maybe a chemical or library of chemicals. These may be cell permeable andtherefore added directly to the cells. Alternatively, it may benecessary to make the cells permeable using streptolysin O, tetanolysinor another permeabilizing agent known by persons skilled in the art. Inanother embodiment, the agent may be a protein or peptide or a nucleicacid sequence. These may be directly applied to the cells, oralternatively introduced into the cells by a vector and when expressed,these proteins and nucleic acids act as intracellular agent. In oneembodiment, agents are nucleic acid sequences are individual DNAmolecules having one or more genes, each operatively linked to the sameor different regulatory sequences to the one operatively linked to thein. The constructs may be introduced simultaneously, or consecutively,each with the same or different markers.

In some embodiments, the cells are exposed to at least one agent. Inother embodiments, the cells are exposed to more than one agent. Theagent may be of the same type (i.e. chemical, procedure, protein,nucleic acid, intrinsic stimuli etc.) or may be any combination ofagents. Exposure of the cells to an agent may be simultaneous,sequentially or consecutive, and in any order. Cells may be exposed toany agent once or any number of times. The time of exposure of the cellsto the agent can vary, depending on the agent and the cells being used,and the level of expression of the nucleic acid construct comprising theBRE and reporter gene.

The cells used in the methods described here in may be prokaryotic, butare preferably eukaryotic, including plant, yeast, worm, insect andmammalian. In another embodiment, the cells are mammalian cells,particularly primate, human and can be associated with any animal ofinterest, including but not limited to, transgenic animals and domesticanimals, such as equine, bovine, murine, ovine, canine, feline, etc.Among these species, various types or cells can be used, such ashematopoetic, neural, mesenchymal, cutaneous, mucosal, stromal, muscle,ovarian, spleen, reticuloendothelial, epithelial, endothelial, hepatic,kidney, gastrointestinal, pulmonary etc. Also, in another embodiment,the cells are stem cells and progenitors, such as hematopoeitic neural,stromal, muscle, ovarian, hepatic, pulmonary, gastrointestinal, etc.

In some embodiments, the reporter gene is any gene which can bedetected. Examples of commonly used reporter genes are fluorescentproteins, for example GFP and GFP-like proteins and modified versionsthereof, and bioluminescent enzymes, for example luciferase or modifiedproteins thereof. Other reporter genes can be chromogenic proteins orenzymes which catalyze a reaction to produce a substrate that isdetectable. Examples of such enzymes include, for example but notlimited to beta-galactosidase, beta-lactamase, beta-glucosidase,beta-glucuronidase, chloramphenicol acetyl transferase. Examples offluorescent proteins are, without limitation green fluorescent protein(GFP) or enhanced green fluorescent protein (EGFP). In alternativeembodiments, the fluorescent protein is yellow fluorescent protein(YFP), an enhanced yellow fluorescent protein (EYFP), a blue fluorescentprotein (BFP), an enhanced blue fluorescent protein (EBFP), a cyanfluorescent protein (CFP), an enhanced cyan fluorescent protein (ECFP)or a red fluorescent protein (dsRED) or any other natural or geneticallyengineered fluorescent protein of those listed above. In yet furtherembodiments, the reconstituted fluorescent proteins may comprise of amixture of fragments from the same or a combination any of the abovelisted fluorescent proteins. Methods to detect the products from thereporter genes are commonly known by persons of ordinary skill in theart.

In some embodiments, the bioluminescent enzyme, for example luciferase,catalyzes a substrate, herein referred to as a “bioluminescencesubstrate” which is a compound that is oxidized in the presence ofluciferase and any necessary activators, to generates light. Thesesubstrates are referred to “luciferins” herein, and are substrates thatundergo oxidation in a bioluminescence reaction. These bioluminescencesubstrates include any luciferin or analogue thereof, or any syntheticcompound that generates light. Such molecules includenaturally-occurring substrates, modified forms thereof and syntheticsubstrates [see e.g. U.S. Pat. Nos. those described in U.S. Pat. Nos.5,374,534 and 5,098,828 which are incorporated herein in their entiretyby reference]. Exemplary luciferins include those described in U.S. Pat.No. 6,436,682, and functional derivatives thereof, analogues thereof,synthetic substrates, as well as dioxetanes [see e.g. U.S. Pat. Nos.5,004,565 and 5,455,357 which are incorporated herein in their entiretyby reference], and other compounds that are oxidized by luciferase in alight-producing reaction [see, e.g. U.S. Pat. Nos. 5,374,534, 5,098,828and 4,950,588 which are incorporated herein in their entirety byreference]. Bioluminescence substrates, thus, include those compoundsthat those skilled in the art recognize as luficerins. In oneembodiment, the luciferin is coelenterazine and analogues thereof, whichinclude molecules in U.S. Pat. No. 6,436,682, and for example, see Zhaoet al, (2004), Mol Imaging, 3;43-54.

Methods to measure bioluminescence are well known to those skilled inthe art. Bioluminescence reactions are also well-known to those skilledin the art, and any such reaction may be adapted for used in combinationwith articles of manufacture as described herein.

The term “agent” refers to any entity which is normally not present ornot present at the levels being administered in the cell. Agent may beselected from a group comprising; chemicals; an action; nucleic acidsequences; proteins; peptides; or fragments thereof. A nucleic acidsequence functioning as an environmental stimuli may be RNA or DNA, andmay be single or double stranded, and can be selected from a groupcomprising; nucleic acid encoding a protein of interest,oligonucleotides, nucleic acids, nucleic acid analogies, for example butnot limited to peptide nucleic acid (PNA), pseudo-complementary PNA(pc-PNA), locked nucleic acids (LNA) and derivatives thereof etc.Nucleic acid sequence encoding environmental stimuli can also inhibitthe activity of a regulatory sequence and/or constitutively activepromoter. Such nucleic acid sequences include, for example, but notlimited to, nucleic acid sequence encoding proteins that act astranscriptional repressors, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but not limited to RNAi,shRNAi, siRNA, micro RNAi (miRNA), antisense oligonucleotides etc. Aprotein and/or peptide or fragment thereof, functioning as anenvironmental stimuli can be any protein of interest, for example, butnot limited to; mutated proteins; therapeutic proteins; truncatedproteins, wherein the protein is normally absent or expressed at lowerlevels in the cell. Proteins of interest can be selected from a groupcomprising; mutated proteins, genetically engineered proteins, peptides,synthetic peptides, recombinant proteins, chimeric proteins, antibodies,midibodies, tribodies, humanized proteins, humanized antibodies,chimeric antibodies, modified proteins and fragments thereof. The agentmay be applied to the media, where it contacts the cell and induces itseffects. Alternatively, the agent may be intracellular within the cellas a result of introduction of the nucleic acid sequence into the celland its transcription resulting in the production of the nucleic acidand/or protein environmental stimuli within the cell.

In some embodiments, the agent is any chemical, entity or moiety,including without limitation synthetic and naturally-occurringnon-proteinaceous entities. In certain embodiments the agent is a smallmolecule having a chemical moiety. For example, chemical moietiesincluded unsubstituted or substituted alkyl, aromatic, or heterocyclylmoieties including macrolides, leptomycins and related natural productsor analogues thereof. Agents can be known to have a desired activityand/or property, or can be selected from a library of diverse compounds.

EXAMPLES

The examples presented herein relate to the use of pyrazoloathrone andfunctional derivatives thereof as a MIS mimetic, and the use ofpyrazoloathrone and functional derivatives thereof for the treatment ofdisease and disorders where MIS is effective at treating or reducing thesymptoms of the disorder. The pyrazoloathrone and functional derivativesthereof can also be used in conjunction with other therapeutic agents,for example chemotherapeutic agents MIS and paclitaxel to reduce theirtherapeutic effective dose in the treatment of cancers. Throughout thisapplication, various publications are referenced. The disclosures of allof the publications and those references cited within those publicationsin their entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains. The following examples are not intendedto limit the scope of the claims to the invention, but are ratherintended to be exemplary of certain embodiments. Any variations in theexemplified methods which occur to the skilled artisan are intended tofall within the scope of the present invention.

Methods

Small molecule screen. To identify molecules that interact with theMISRII, we developed a reporter assay where binding to MISRII activatesa firefly luciferase reporter driven by the BMP-responsive element (BRE)promoter kindly provided by Dr. Peter ten Dijke (The Netherlands CancerInstitute, The Netherlands) The BMP promoter region on mouse M. mus islocated on chromosome 2 (Chr2) and corresponds to RefSeq No:NT_(—)039207.6 93504644-93506272 (SEQ ID NO:1). The BRE-luciferasereporter (Korchynskyi and ten Dijke, 2002, Logeart-Avramoglou, 2006) hasmultiple optimized BMP response elements (3 SBE, 7 GC, and 4 CAGC)driving the expression of the firefly luciferase. This reporterconstruct together with a control Renilla luciferase reporter phRL-CMV(Promega, Madison Wis.) and a rat MISRII cDNA expression construct(Teixeira, 1996) were transiently transfected into COS7 cells and platedin 384 well plates. Primary screens were performed at the HarvardInstitute of Chemistry and Cell Biology screening facility. Compoundsfrom chosen libraries were added at a volume of 100 nl at 5 mg/mlconcentration in DMSO into 30 μl cell media and cultured for 24 h at 37°C., after which the luciferase activity was measured with reagents fromPromega according to the manufacturer's instructions. A hit wasidentified as causing a fold induction over the plate median and eachcompound was tested in duplicate in separate plates. MIS was used tocontrol for intra- and inter-assay variability.

Cell culture. COS7 cells were cultured in DMEM supplemented with 10%female Fetal Bovine Serum (FBS) and MOVCAR7 cells were maintained inDMEM with 4% female FBS. Both cell-lines were cultured in a humidified5% CO₂ incubator at 37° C. with added 2 mM L-glutamine, 100 U/mlpenicillin and 100 μg/ml streptomycin.

Transfection and reporter assay. COS7 cells were plated at a density of5000 cells/well in 96-well plates for secondary screens. The day afterplating cells were transfected with rat MISRII receptor or EGFP-N1(Clontech, Mountain View Calif.) together with BRE firefly luciferasereporter construct and phRL-CMV using TransIT-LT1 transfection reagent(Mirus Bio Corporation, Madison Wis.). The following day, cells weretreated with SP600125 and/or MIS at the indicated concentrations for 24h, or as otherwise stated for the time course experiments, in 1% serummedia. MOVCAR7 cells were transfected with the BRE and phRL-CMVluciferase reporters and treated with MIS and/or SP600125 at theindicated concentrations in growth media for 24 h. Results were obtainedusing the Dual Luciferase reporter assay according to the manufacturer'sinstructions (Promega). Plates were read in a Wallac Victor2 luminometer(PerkinElmer, Wellesley Mass.). BMP2 and activin were purchased from R&DSystems, Minneapolis Minn.

Proliferation assay. The effect of SP600125 on cell proliferation wasmeasured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay (Mosmann, 1983) in a mouse ovarian cancer cell line,MOVCAR7 cells that were kindly provided by Dr. Denise Connolly, FoxChase Cancer Center, Philadelphia Pa. (Connolly, 2003). The 4306 cellswere provided by Dr. Daniela Dinulescu, Brigham and Women's (Boston)(30). Cells were plated at a density of 1000 cells/well in 96-wellplates and treated 24 h later. The cell proliferation was measured after7 days by registering the absorbance at 550 nm. Paclitaxel wasmanufactured by Ivax Pharmaceuticals and obtained from the MGH pharmacy.

Western Blotting. Cells were washed in PBS and lysates were prepared in1× Passive lysis buffer (PLB) provided with the Dual luciferase reporterassay kit (Promega). The lysates were cleared by centrifugation and theprotein concentration was determined by Bradford assay (Bio-Rad,Hercules Calif.). Equal volumes of protein were loaded onto a NuPAGE4-12% Bis-Tris gradient gel with MOPS-buffer (Invitrogen, CarlsbadCalif.) for polyacrylamide gel electrophoresis. Following transfer to anitrocellulose by electroblotting, the membrane was blocked in 5% milkTris-buffered saline/Tween20 (TBS-T) for 1 h at room temperature andincubated with primary antibody (pJNK, c-jun and phosph c-jun antibodieswere purchaced from Cell Signalling, Danvers, Mass.; amd JNK antibodywas published from Santa Cruz Biotechnology, CA) overnight at 4° C. in5% BSA followed by incubation with secondary horseradish peroxidaseconjugated anti-rabbit antibody (Jackson ImmunoResearch, Westgrove Pa.)for 1 h. Immunoreactive proteins were detected by enhancedchemiluminescence (Pierce, Rockford Ill.). Anisomycin was purchased fromCalbiochem.

MIS preparation and bioassay. Recombinant human MIS was prepared fromChinese hamster ovary cells stably transfected with a construct of thehuman MIS gene and purified from serum-free media by serial carbohydrateaffinity and ion exchange chromatography or by immunoaffinitychromatography (Lorenzo, 2002). The bioactivity of the MIS was thenverified using an established organ culture assay, which gradesMüllerian duct regression of the female 14.5-day gestation raturogenital ridge (Donahoe, 1977).

Statistical analysis. Data were analyzed by ANOVA of the repeatedexperiments followed by the Tukey's posthoc test when appropriate withPrism (GraphPad Software, San Diego Calif.). When the control data wasnormalized to 1, this data was excluded from the statistical analyses.Synergy was calculated by two-way factorial ANOVA (Pieretti-Vanmarcke,2006a). For all analyses, significance was assigned at p≦0.05.

Example 1

SP600125 activates a BMP-responsive promoter in conjunction with MISRII.MIS-specific signaling absolutely requires the MIS type II receptor andknockout studies in mice show phenocopy results when either MISRII orthe MIS ligand is deleted (Behringer, 1994, Mishina, 1996). This allowedthe inventors to develop an exquisitely precise screen for MIS signalingso that only cells that express MISRII initiate MIS downstreamsignaling. The inventors used the BRE-luciferase reporter in COS cellscotransfected with an expression vector containing the rat MISRIIreceptor cDNA (Teixeira, 1996). Screening of 15,000 compounds resultedin several hits that activated the MISRII at levels near those caused byits native ligand, MIS, in a reporter gene assay that identifiescompounds that activate the BMP-responsive element through MISRIIinteraction. The first suitable candidate to undergo further analysisafter the primary screen was the pyrazoloathrone derivative;antra[1,9-cd]pyrazol-6(2H)-one, a JNK inhibitor II, also known asSP600125.

To characterize the activity of SP600125 and its effect on MIS signaltransduction, the inventors performed experiments with increasingconcentrations of SP600125 and measured BRE-luciferase activity (FIG.1). SP600125 induced luciferase expression in a concentration-dependentmanner in MISRII-transfected COS7 cells and at 25 uM reached 6-foldhigher than that of vehicle-treated cells. Luciferase expression wasalso induced with 33.3 uM and 100 uM concentrations of added SP600125 by15- and 25-fold, respectively, (shown with bars after the dotted line)but the Renilla luciferase was inhibited by approximately 60% suggestingtoxic effects to the cells. Only cells transfected with MISRII showed asignificant induction in luciferase activity indicating that MISRIIexpression was necessary for SP600125 to induce MIS signaling in thesecells.

To further demonstrate that the activating effect of SP600125 onBRE-luciferase was MISRII specific, the inventors used a reporterconstruct where a conserved lysine (K228) in the ATP-binding region ofthe receptor was changed to an arginine (K228R), disabling the kinaseactivity of the receptor (4). In FIG. 5 the inventors demonstrate thatneither MIS nor SP600125 activates BRE-luciferase when kinase-deficientreceptor is overexpressed. In addition, the inventors demonstrated thata further increase of the activity when MUS was added in combinationwith SP600125, an effect that was completely abolished with the K228R,demonstrating that the serine/theronine activity of the MISRII is neededfor activation of BRE-luciferase.

Little is known about the down-stream signaling events that follow MISbinding to its receptor. Reporters that have been used to study MISeffects include the BMP-specific reporter genes XVent2 and Tlx (Clarke,2001b). In this study the inventors used a BMP-responsive (BRE) promoterto drive expression of firefly luciferase. This reporter is alsoactivated by BMPs, but not by TGFβ or activin (Korchynskyi and tenDijke, 2002). To investigate the receptor-specificity of SP600125 andexclude the possibility that this molecule activates other signalingpathways common for the TGFβ family, the inventors transfected COS7cells with a BMP type II receptor (BMPRII) expression construct and theBRE-luciferase reporter. As expected, 25 ng/ml BMP2, a ligand forBMPRII, induced BRE-luciferase activity in cells transfected withBMPRII. However, in contrast to the synergistic induction observed withthe MISRII-transfected cells treated with MIS and SP600125, cotreatmentof BMPRII-transfected cells with BMP2 and 10 μM SP600125 decreasedluciferase activity by 25% compared to BMP2 alone (FIG. 2D). Inaddition, the inventors tested whether the type II receptors for TGFβand activin responded to SP600125 by co-transfecting the correspondingreceptor with a CAGA-promoter driven luciferase reporter into COS7cells. This reporter construct has been shown to be activated by bothactivin and TGFβ via the smad3 pathway (Dennler, 1998). However,SP600125 did not have any significant effect alone or in combinationwith the receptor-ligand (data not shown).

Example 2

Synergy with SP600125 and MIS. In the next set of experiments, theinventors assessed whether addition of MIS and SP600125 resulted in anadditive or cooperated to provide a synergistic effect on BRE-luciferaseactivity. COS7 cells were cotransfected with the luciferase reportersand the MISRII expression construct, treated with a combination of MISand SP600125 and compared to cells treated with MIS alone. In FIG. 2A,the inventor discovered that MIS added at a final concentration of 0.5ug/ml induced luciferase expression greater than ten-fold. Addition ofas little as 5 uM SP600125 increased the MIS-mediated induction byapproximately 16-fold, equivalent to an additional six-fold over MISalone. Increasing the concentration of SP600125 to 10 uM and 25 uMresulted in inductions of 22- and 25-fold, respectively. Statisticalanalyses by two-way ANOVA showed a synergistic effect on the activationof BRE-luciferase when MIS and SP600125 were combined compared witheither alone. Addition of a different small molecule inhibitor of JNK,JNK inhibitor VIII, did not result in any activation of BRE-luciferaseat 5, 25 or 50 μM concentration and no further effect was seen whencombined with MIS (FIG. 2B). Additionally, a peptide inhibitor, JNKinhibitor III, did not result in any activation of BRE-luciferase at 1or 5 μM concentration and no further effect was seen when combined withMIS (data not shown). The synergy of SP600125 and MIS was also tested ina time course experiment. In FIG. 2C, the inventors discovered that 10uM SP600125 and 0.5 ug/ml MIS maintain their synergistic effect onluciferase activity after 6 h through 48 h co-treatment over that ofeach treatment alone.

In order to rule out the possibility that BRE-Luciferase induction bySP600125 might be an artifact of MISRII overexpression in COS7 cells,the inventors transfected the luciferase reporter constructs into amouse ovarian cancer cell line, MOVCAR7, that expresses endogenousMISRII (Pieretti-Vanmarcke, 2006b), and assayed for their response toMIS and SP600125. Treatment of the cells with 5 μg/ml MIS resulted in a2-fold increase of BRE activity compared to untreated cells. Treatmentwith 25 μM SP600125 resulted in a 5-fold increase in BRE-luciferaseactivity compared to untreated control. The combined treatment with MISand SP600125 gave a 16-fold increase in the BRE-response, showing asynergistic effect with more than twice the activity of each treatmentalone, which was similar to that observed in COS7 cells (FIG. 2E).

Example 3

MIS does not activate the JNK pathway. In order to test the possibilitythat the effects observed with SP600125 are the result of an indirecteffect of MISRII-mediated activation of the JNK-pathway the inventorstreated MOVCAR7 cells with 5 μg/ml MIS and measured phosphorylation ofJNK by immunoblotting (FIG. 3A). The known Jun kinase activatoranisomycin was used as a positive control (Hazzalin, 1998). However, nophosphorylation beyond background was observed after 30 min MIStreatment, although a strong activation was achieved with 500 nManisomycin. Further, MIS had no effect on phosphorylation of JNK byanisomycin. To examine the effects of SP600125 on phosphorylation ofc-jun, a downstream target for phosphorylation by JNK, the inventorspre-treated MOVCAR cells with SP600125 before treatment with anisomycinand assessed phospho-c-jun by western blot analysis (FIG. 3B). While theinventors observed inhibition of c-jun phosphorylation with 25 uMSP600125, at 10 uM SP600125, twice the concentration at which synergywith MIS and paclitaxel occurred (see below), the inventors did observemarginal and inconsistent inhibition of c-jun phosphorylation andtherefore the inventors have discovered that the effects observed arenot due to SP600125-mediated inhibition of the JNK signaling pathway.

Example 4

SP600125 inhibits proliferation of an ovarian cancer cell lines. MISinhibits both the proliferation of MOVCAR7 cells in vitro inproliferation assays and tumor growth in vivo when these cells arexenotransplanted into an immunocompromised mouse model(Pieretti-Vanmarcke, 2006b). Therefore, the inventors tested whetherSP600125 had similar effects on MOVCAR7 proliferation in vitro againusing the cell proliferation assay. The cells were treated for 7 daysand the proliferation was measured by spectrophotometry. To excludecytotoxic effects on the cells unrelated to MIS signal transduction, weused untransfected COS7 cells as a negative control. The results, asshown in FIG. 4A indicate that SP600125 inhibited cell proliferation inMOVCAR cells in a similar manner to MIS (Pieretti-Vanmarcke, 2006a,Pieretti-Vanmarcke, 2006b) and to a greater extent than in untransfectedCOS7 cells (IC50 4.5 μM vs. 9.5 μM, respectively). The inventors alsoinvestigated whether SP600125 could potentiate the effect of an agentalready established in the treatment of ovarian cancer, paclitaxel (FIG.4B). Paclitaxel is used in treatment of a wide range of tumors, but theside effects can be debilitating (Pieretti-Vanmarcke, 2006a,Pieretti-Vanmarcke, 2006b). The inventors used 2 μM and 5 μM of SP600125with paclitaxel since these doses inhibited MOVCAR7 cells, but hadminimal effect on COS7 cells of only 20% (FIG. 4A). Combining 5 μM ofSP600125 with increasing doses of paclitaxel significantly shifted thecurve to the left as compared with the paclitaxel alone (IC₅₀ 0.21 nMvs. 0.81 nM), demonstrating that adjuvant therapy with SP600125 can bebeneficial for patients undergoing treatment with paclitaxel.

The inventors also tested whether SP600125 could inhibit theproliferation of another mouse ovarian cancer cell line that isinhibited by MIS, 4306 cells (Pieretti-Vanmarcke, 2006a,Pieretti-Vanmarcke, 2006b). In FIG. 4C, the inventors demonstrate thatSP600125 at a concentration of 2 μM inhibited proliferation of 4306cells by 50%. When combined with paclitaxel, SP600125 exerted asynergistically significant inhibition of 4306 proliferation over thatof paclitaxel alone. If 2 μM SP600125 is combined with MIS or with MISand paclitaxel, the levels of cell proliferation is also significantlyinhibited and with synergy over MIS alone. These results with anotherMIS-responsive ovarian cancer cell line demonstrate that SP600125 isalso beneficial when MIS-paclitaxel combination therapy becomesavailable.

Example 5

The growing evidence showing that MIS can act as an inhibitor of tumorcell growth and as a potential drug in the treatment of ovarian cancerlead the inventors to screen for a small molecule that could mimic thereceptor-mediated effects of MIS for use either alone or in combinationwith MIS or other chemotherapeutic agents. From this screening, theinventors discovered SP600125 as a small molecule activator of the MIStype II receptor signaling and a possible candidate for a drug withMIS-like effects. The inventors have discovered that SP600125 activatessignaling downstream of the MISRII in a receptor-dependent manner andthat it has a synergistic effect on the cellular response to MIS.

With the growing evidence showing that MIS can act as an inhibitor oftumor cell growth and as a potential therapeutic treatment for ovariancancer, the inventors screened for a small molecule that could mimic thereceptor-mediated effects of MIS for use either alone or in combinationwith MIS or other chemotherapeutic agents. In this screening, theinventors identified SP600125 as a small-molecule activator of theMISRII signaling that had MIS-like effects. As disclosed herein, theinventors have demonstrated that SP600125 activates signaling downstreamof the MISRII in a receptor-dependent manner that is dependent on thekinase activity of the MISRII and that it has a synergistic effect onthe cellular response to MIS. A role for the JNK pathway in MISRIIsignaling has not yet been reported, and the inventors did not observeany phosphorylation of JNK by immunoblotting after treatment of cellswith MIS.

To exclude the possibility that JNK signaling pathway exerts a negativeeffect on MISRII signaling, the inventors demonstrated that otherinhibitors to JNK, JNK inhibit or VIII, which is also a reversibleATP-competitive inhibitor of JNK(41), and JNK inhibitor III, a junpeptide inhibitor, had no effect on activation of the BRE-luciferasewith or without added MIS. Furthermore, JNK inhibitor VIII is much moreeffective in inhibiting phosphorylation of c-jun than is SP600125,indicating that it would have the same or greater effect on activationof MISRII-mediated signaling if the effect was mainly through inhibitionof JNK. The inventors therefore discovered that SP600125 modifies MISRIIsignal transduction by interacting with the receptor or other moleculesnecessary for the activation of the receptor.

The inventors also demonstrate herein that SP600125 activates MISRIIsignaling in a concentration-dependent manner. This effect is specificfor the MISRII and was not seen with cells that did not express MISRIIor with transfection of other receptors of the TGFβ family. Preliminarystudies also demonstrate that that canonical Smad1/5/8 phosphorylationis not involved in SP600125-mediated signaling (data not shown).

Based on specificity which was demonstrated in the reporter assay usingCOS7 cells overexpressing the receptors, the inventors next examined theeffect of SP600125 on the proliferation of ovarian cancer cell linesexpressing the MISRII. Using MOV-CART cells in the reporter assaydemonstrated a similar response pattern and was not an artifact due toMISRII overexpression. The inventors also demonstrated that SP600125inhibited the proliferation of MOVCAR7 cells to a significantly higherdegree than it did in untransfected COS7 cells, demonstrating thatendogenous MISRII functions as target for SP600125, and that targetedtherapeutics which target MISRII are useful to minimize cytotoxic sideeffects. The inhibitory effect of SP600125 on ovarian cancer cellproliferation was also observed in 4306 cells, another MIS-responsivecell line (Pieretti-Vanmarcke et al, 2006; Proc Natl Acad Sci USA103:17426-1743116; Pieretti-Vanmarcke et al, 2006; Clin Cancer Res12:1593-1598).

As disclosed herein, the inventors demonstrate that the inhibitoryeffects of SP600125 on ovarian cancer cells is mediated by MISRIIsignaling, and that SP600125 activates the BRE-luciferase reporter inMISRII-expressing cells (FIG. 2E), an effect that is synergistic withMIS. Additionally, the inventors discovered that MIS and SP600125together had a greater effect on 4306 proliferation than with MIS alone(FIG. 4C). Taken together, the inventors have discovered that theinhibitory effect of SP600125 on ovarian cancer cell proliferationoccurs in a manner similar to that observed with MIS (Pieretti-Vanmarckeet al, 2006; Proc Natl Acad Sci USA 103:17426-1743116;Pieretti-Vanmarcke et al, 2006; Clin Cancer Res 12:1593-1598), and theinduced response of the BRE-luciferase reporter in MIS-expressing cellsdemonstrated that SP600125 is affecting ovarian cancer cellproliferation at least in part by activating MISRII signaling.

As SP600125 has been shown to inhibit the proliferation of humanendothelial and prostate tumor cell lines both in vitro by arresting thecells in G2/M phase of the cell cycle and in vivo by inhibiting thegrowth of injected tumor cells and increasing the efficacy ofcyclophosphamide treatment (Ennis et al., 2005;.) Pharmacol Exp Ther313:325-332). Combined with the demonstration of inhibition ofMIS-expressing cancer cells as disclosed herein, SP600125 may bebeneficial for the treatment of a variety of other cancers as well.

Paclitaxel has been used in the treatment of a wide range of tumors,including ovarian, breast, and prostate. It exerts its effect bytargeting the microtubules and arresting the cells in G2/M phase,leading to the induction of apoptosis (Bhalla et al, 2003, Oncogene22:9075-908645). In contrast to the inventor's discoveries as disclosedherein, previous reports have shown that SP600125 reduced the apoptoticeffect of paclitaxel on prostate cancer cells in vitro (Zhang et al.,2006, Int J Cancer 118:2072-208146) and in ovarian cancer cells (Wang etal, 2006; Oncogene 25:4857-486647). Therefore, one would not expectSP600125 to increase the effect of paclitaxel on cancer cells, such asMISRII expressing cancer cells as disclosed herein. Furthermore, theinventors used lower doses of paclitaxel than the previous reports incombination with SP600125 to increase the effect paclitaxel. In fact,the inventors demonstrate that the effect of combination of paclitaxeland SP600125 treatment was optimized at a lower concentration ofpaclitaxel. Furthermore, the inventors demonstrated that lowerconcentrations of SP600125, 2-5 μM as compared with the 10-20 μMconcentrations did not result in apoptosis. Additionally, paclitaxel isthought to exert a biphasic apoptosis effect through a p53-dependentpathway that induces apoptosis at lower concentrations, whereas athigher concentrations, it inhibits p53 expression in an a sopharyngealcarcinoma cell line (Tan et al., 2002; Int J Cancer 97:168-172). Thesedata demonstrate that a concentration-dependent difference in apoptosismediated by SP600125 alone or in combination with paclitaxel may beoperative in different cells or tissues.

By using different inhibitors to JNK, a c-jun peptide inhibitor or JNKinhibitor III (Calbiochem) and another small molecule inhibitor, JNKinhibitor VIII, the inventors excluded the possibility of JNK exerting anegative effect on MISRII signaling. These inhibitors had no effect onactivation of the BRE-luciferase by itself or in combination with MIS,indicating that the inventors have discovered that SP600125 modifiesMISRII signal transduction not via interacting with JNK but byinteracting with the receptor or other molecules necessary for theactivation of the receptor.

Furthermore, the inventors have discovered that SP600125 activatesMISRII signaling in a concentration-dependent manner, and alsodiscovered that SP600125 is specific for the MIS type II receptor, anddoes not activate MISRII signaling in cells not expressing MISRII orwith transfection of other receptors of the TGFβ family.

The inventors also discovered that SP600125 inhibited the proliferationof MOVCAR7 and 4307 cells, both ovarian cancer lines expressing MISRIIto a significantly higher degree than it did in COST cells,demonstrating that endogenous MISRII is a target for pyrazoloathronesand analogues, for example SP600125.

In addition, the inventors have surprisingly discovered that SP600125lowers the concentration of paclitaxel needed for an apoptotic effect.Paclitaxel has been used in the treatment of a wide range of tumors,including ovarian, breast, and prostate. It exerts its effect bytargeting the microtubules and arresting the cells in G2/M-phase,leading to the induction of apoptosis (Bhalla, 2003). In fact, theinventors have discovered that in combination with SP600125, the optimalconcentration of paclitaxal is much lower than the concentration ofpaclitaxel used alone. This is a surprising discovery and in starkcontrast with other findings, where SP600125 was shown to reduce theapoptotic effect of paclitaxel on prostate cancer cells in vitro (Zhang,2006) and in ovarian cancer cells (Wang, 2006).

In conclusion, the inventor have discovered a small molecule SP600125that has the same biological activity and functions as MIS, and resultsin MIS receptor-mediated downstream effects. Accordingly, the inventorshave demonstrated that SP600125 can result in inhibition of tumor cellproliferation, and can be used as a therapeutic agent in treatment ofMISRII-positive tumors. As disclosed herein, the inventors havediscovered that SP600125, a JNK inhibitor, is a small molecule that canmimic the effects of MIS in a receptor-specific manner. The inventorshave also discovered that SP600125 can lower the effective concentrationof MIS and can be used as a therapeutic for the treatment of ovariancancer. The inventors have also demonstrated that SP600125 can functionin concert to lower the effective therapeutic dose of alreadyestablished anti-cancer therapeutic agents such as paclitaxel, as wellas MIS.

REFERENCES

The references cited herein and throughout the application areincorporated herein in their entirety by reference.

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SEQUENCES

SEQ ID NO: 1: NT_039207.6 93504644-93506272 SEQ ID NO: 2: NM_000479    1gcatgttgac acatcaggcc cagctctatc actggggagg gagataggct gccagggaca   61gaaagggctc tttgagaagg ccactctgcc tggagtgggg gcgccgggca ctgtccccca  121aggtcgcggc agaggagata ggggtctgtc ctgcacaaac accccacctt ccactcggct  181cacttaaggc aggcagccca gcccctggca gcacccacga tgcgggacct gcctctcacc  241agcctggccc tagtgctgtc tgccctgggg gctctgctgg ggactgaggc cctcagagca  301gaggagccag ctgtgggcac cagtggcctc atcttccgag aagacttgga ctggcctcca  361ggcagcccac aagagcctct gtgcctggtg gcactgggcg gggacagcaa tggcagcagc  421tcccccctgc gggtggtggg ggctctaagc gcctatgagc aggccttcct gggggccgtg  481cagagggccc gctggggccc ccgagacctg gccaccttcg gggtctgcaa caccggtgac  541aggcaggctg ccttgccctc tctacggcgg ctgggggcct ggctgcggga ccctgggggg  601cagcgcctgg tggtcctaca cctggaggaa gtgacctggg agccaacacc ctcgctgagg  661ttccaggagc ccccgcctgg aggagctggc cccccagagc tggcgctgct ggtgctgtac  721cctgggcctg gccctgaggt cactgtgacg agggctgggc tgccgggtgc ccagagcctc  781tgcccctccc gagacacccg ctacctggtg ttagcggtgg accgccctgc gggggcctgg  841cgcggctccg ggctggcctt gaccctgcag ccccgcggag aggactcccg gctgagtacc  901gcccggctgc aggcactgct gttcggcgac gaccaccgct gcttcacacg gatgaccccg  961gccctgctcc tgctgccgcg gtccgagccc gcgccgctgc ctgcgcacgg ccagctggac 1021accgtgccct tcccgccgcc caggccatcc gcggaactcg aggagtcgcc acccagcgca 1081gaccccttcc tggagacgct cacgcgcctg gtgcgggcgc tgcgggtccc cccggcccgg 1141gcctccgcgc cgcgcctggc cctggatccg gacgcgctgg ccggcttccc gcagggccta 1201gtcaacctgt cggaccccgc ggcgctggag cgcctactcg acggcgagga gccgctgctg 1261ctgctgctga ggcccactgc ggccaccacc ggggatcctg cgcccctgca cgaccccacg 1321tcggcgccgt gggccacggc cctggcgcgc cgcgtggctg ctgaactgca agcggcggct 1381gccgagctgc gaagcctccc gggtctgcct ccggccacag ccccgctgct ggcgcgcctg 1441ctcgcgctct gcccaggtgg ccccggcggc ctcggcgatc ccctgcgagc gctgctgctc 1501ctgaaggcgc tgcagggcct gcgcgtggag tggcgcgggc gggatccgcg cgggccgggt 1561cgggcacagc gcagcgcggg ggccaccgcc gccgacgggc cgtgcgcgct gcgcgagctc 1621agcgtagacc tccgcgccga gcgctccgta ctcatccccg agacctacca ggccaacaat 1681tgccagggcg tgtgcggctg gcctcagtcc gaccgcaacc cgcgctacgg caaccacgtg 1741gtgctgctgc tgaagatgca ggcccgtggg gccgccctgg cgcgcccacc ctgctgcgtg 1801cccaccgcct acgcgggcaa gctgctcatc agcctgtcgg aggagcgcat cagcgcgcac 1861cacgtgccca acatggtggc caccgagtgt ggctgccggt gacccctgcg ccgcgcggac 1921tcctgccccg agggtccgga cgcgccccag ctcgcgcccc ttcccatatt tattcggacc 1981ccaagcatcg ccccaataaa gaccagcaag caaccggcaa aaaaaaaaaa aaaaaaaaaa 2041aaaaaaaaaa aaaaaaaaaa aaaaa SEQ ID NO: 3 KO3474    1 cacatcaggcccagctctat cactggggag ggagataggc tgccagggac agaaagggct   61 ctttgagaaggccactctgc ctggagtggg ggcgccgggc actgtccccc aaggtcgcgg  121 cagaggagataggggtctgt cctgcacaaa caccccacct tccactcggc tcacttaagg  181 caggcagcccagcccctggc agcacccacg atgcgggacc tgcctctcac cagcctggcc  241 ctagtgctgtctgccctggg ggctctgctg gggactgagg ccctcagagc agaggagcca  301 gctgtgggcaccagtggcct catcttccga gaagacttgg actggcctcc aggcatccca  361 caagagcctctgtgcctggt ggcactgggc ggggacagca atggcagcag ctcccccctg  421 cgggtggtgggggctctaag cgcctatgag caggccttcc tgggggccgt gcagagggcc  481 cgctggggcccccgagacct ggccaccttc ggggtctgca acaccggtga caggcaggct  541 gccttgccctctctacggcg gctgggggcc tggctgcggg accctggggg gcagcgcctg  601 gtggtcctacacctggagga aggtatgtgg ggcccagccc caagcttggc accgccgtct  661 tccttcaggtgggccgggtc ctcctaggga agatcagggg ctggcagagc ccccaccctg  721 ggcagggaggctgtggtctt gttcctagga ctgggttgcg ggtccgtggc ctggaaggtg  781 ggcaccacactctgtcctgt ccccgaagcc cagctcttag acttgcccct gcctcggtgc  841 cagggagagagctgctgcct tctccccacc cctgaagacg acgcagggct cggggccagt  901 ggaacccttcttcccacagc cccagcctgt tctcagggcc gctggcctaa gatactccct  961 gcggggaaggggcttcatcg ggcaccccaa cccagagacc ccagggcggc agccccaccc 1021 acagcctcagacgcagcccc tgcctgcccc tgccgtcacc gctccctggc tgcaggaagg 1081 cagctaagaggggcaccctt gtcccccgct tgaggtcccc tgcacagtgg ccagagcggc 1141 agggacagatcccaaagatt cccggggggt gtggccttca atggctcagg cgtcccctgc 1201 tgtcccggctgcagtgacct gggagccaac accctcgctg aggttccagg agcccccgcc 1261 tggaggagctggccccccag agctggcgct gctggtgctg taccctgggc ctggccctga 1321 ggtcactgtgacgagggctg ggctgccggg tgcccaggta ccagggagtt gcatggggca 1381 gtgcccgggccgtggcgggg ggcatgaatt tgttgcaggg tctgcagtac tgagaacagc 1441 gtagaaccagtggcgatggg aggaagggga ccggtagagc ggggctgggt aagcctccat 1501 ccagccgggctgagccctgg tctccgcaga gcctctgccc ctcccgagac acccgctacc 1561 tggtgttagcggtggaccgc cctgcggggg cctggcgcgg ctccgggctg gccttgaccc 1621 tgcagccccgcggagagggt aggtccgcgt ggagagggac ggggagccgg gtcgactgcc 1681 cccgggcccccagcccctga gccagccgcg tgcccaccca ccgcagactc ccggctgagt 1741 accgcccggctgcaggcact gctgttcggc gacgaccacc gctgcttcac acggatgacc 1801 ccggccctgctcctgctgcc gcggtccgag cccgcgccgc tgcctgcgca cggccagctg 1861 gacaccgtgcccttcccgcc gcccaggtgc gcgcaggcac cgggacacgg ggcaggagcg 1921 ggcgggggcggcgtggcctc gtggccgctc tcaactcctc caattgcggg ttccaggcca 1981 tccgcggaactcgaggagtc gccacccagc gcagacccct tcctggagac gctcacgcgc 2041 ctggtgcgggcgctgcgggt ccccccggcc cgggcctccg cgccgcgcct ggccctggat 2101 ccggacgcgctggccggctt cccgcagggc ctagtcaacc tgtcggaccc cgcggcgctg 2161 gagcgcctactcgacggcga ggagccgctg ctgctgctgc tgaggcccac tgcggccacc 2221 accggggatcctgcgcccct gcacgacccc acgtcggcgc cgtgggccac ggccctggcg 2281 cgccgcgtggctgctgaact gcaagcggcg gctgccgagc tgcgaagcct cccgggtctg 2341 cctccggccacagccccgct gctggcgcgc ctgctcgcgc tctgcccagg aggccccggc 2401 ggcctcggcgatcccctgcg agcgctgctg ctcctgaagg cgctgcaggg cctgcgcgtg 2461 gagtggcgcgggcgggatcc gcgcgggccg ggtcgggcac agcgcagcgc gggggccacc 2521 gccgccgacgggccgtgcgc gctgcgcgag ctcagcgtag acctccgcgc cgagcgctcc 2581 gtactcatccccgagaccta ccaggccaac aattgccagg gcgtgtgcgg ctggcctcag 2641 tccgaccgcaacccgcgcta cggcaaccac gtggtgctgc tgctgaagat gcaggcccgt 2701 ggggccgccctggcgcgccc accctgctgc gtgcccaccg cctacgcggg caagctgctc 2761 atcagcctgtcggaggaacg catcagcgcg caccacgtgc ccaacatggt ggccaccgag 2821 tgtggctgccggtgacccct gcgccgcgcg gactcctgcc ccgagggtcc ggacgcgccc 2881 cagctcgcgccccttcccat atttattcgg accccaagca tcgccccaat aaagaccagc 2941 aagcaaccggctggggtgtc cgtgcgtgtt agggggcccg tgggacctcc cttgccgtct 3001 ctcctcgcgcacggcccggg tccgccctgt agcgctcgct gtctctcccc tgcctgaagc 3061 gccccaccaccgtctttcag gccccggact tggtgccggg SEQ ID NO: 4 NM_020547    1 atctgaagaaagatttggcc aggggcagct gtgctggctt atgctcttct ccttctgctg   61 ctgccatcctccagcaagat gctagggtct ttggggcttt gggcattact tcccacagct  121 gtggaagcacccccaaacag gcgaacctgt gtgttctttg aggcccctgg agtgcgggga  181 agcacaaagacactgggaga gctgctagat acaggcacag agctccccag agctatccgc  241 tgcctctacagccgctgctg ctttgggatc tggaacctga cccaagaccg ggcacaggtg  301 gaaatgcaaggatgccgaga cagtgatgag ccaggctgtg agtccctcca ctgtgaccca  361 agtccccgagcccaccccag ccctggctcc actctcttca cctgctcctg tggcactgac  421 ttctgcaatgccaattacag ccatctgcct cctccaggga gccctgggac tcctggctcc  481 cagggtccccaggctgcccc aggtgagtcc atctggatgg cactggtgct gctggggctg  541 ttcctcctcctcctgctgct gctgggcagc atcatcttgg ccctgctaca gcgaaagaac  601 tacagagtgcgaggtgagcc agtgccagag ccaaggccag actcaggcag ggactggagt  661 gtggagctgcaggagctgcc tgagctgtgt ttctcccagg taatccggga aggaggtcat  721 gcagtggtttgggccgggca gctgcaagga aaactggttg ccatcaaggc cttcccaccg  781 aggtctgtggctcagttcca agctgagaga gcattgtacg aacttccagg cctacagcac  841 gaccacattgtccgatttat cactgccagc cgggggggtc ctggccgcct gctctctggg  901 cccctgctggtactggaact gcatcccaag ggctccctgt gccactactt gacccagtac  961 accagtgactggggaagttc cctgcggatg gcactgtccc tggcccaggg cctggcattt 1021 ctccatgaggagcgctggca gaatggccaa tataaaccag gtattgccca ccgagatctg 1081 agcagccagaatgtgctcat tcgggaagat ggatcgtgtg ccattggaga cctgggcctt 1141 gccttggtgctccctggcct cactcagccc cctgcctgga cccctactca accacaaggc 1201 ccagctgccatcatggaagc tggcacccag aggtacatgg caccagagct cttggacaag 1261 actctggacctacaggattg gggcatggcc ctccgacgag ctgatattta ctctttggct 1321 ctgctcctgtgggagatact gagccgctgc ccagatttga ggcctgacag cagtccacca 1381 cccttccaactggcctatga ggcagaactg ggcaataccc ctacctctga tgagctatgg 1441 gccttggcagtgcaggagag gaggcgtccc tacatcccat ccacctggcg ctgctttgcc 1501 acagaccctgatgggctgag ggagctccta gaagactgtt gggatgcaga cccagaagca 1561 cggctgacagctgagtgtgt acagcagcgc ctggctgcct tggcccatcc tcaagagagc 1621 cacccctttccagagagctg tccacgtggc tgcccacctc tctgcccaga agactgtact 1681 tcaattcctgcccctaccat cctcccctgt aggcctcagc ggagtgcctg ccacttcagc 1741 gttcagcaaggcccttgttc caggaatcct cagcctgcct gtaccctttc tcctgtgtaa 1801 atatgcagtttatgtgtcat caatgtacat gccaacataa atatggcgat tgtat SEQ ID NO: 5 AF172932   1 ggcacgaggg cagctgtgct ggcttatgct cttctccttc tgctgctgcc atcctccagc  61 aagatgctag ggtctttggg gctttgggca ttacttccca cagctgtgga agcaccccca 121 aacaggcgaa cctgtgtgtt ctttgaggcc cctggagtgc ggggaagcac aaagacactg 181 ggagagctgc tagatacagg cacagagctc cccagagcta tccgctgcct ctacagccgc 241 tgctgctttg ggatctggaa cctgacccaa gaccgggcac aggtggaaat gcaaggatgc 301 cgagacagtg atgagccagg ctgtgagtcc ctccactgtg acccaagtcc ccgagcccac 361 cccagccctg gctccactct cttcacctgc tcctgtggca ctgacttctg caatgccaat 421 tacagccatc tgcctcctcc agggagccct gggactcctg gctcccaggg tccccaggct 481 gccccaggtg agtccatctg gatggcactg gtgctgctgg ggctgttcct cctcctcctg 541 ctgctgctgg gcagcatcat cttggccctg ctacagcgaa agaactacag agtgcgaggt 601 gagccagtgc cagagccaag gccagactca ggcagggact ggagtgtgga gctgcaggag 661 ctgcctgagc tgtgtttctc ccaggtaatc cgggaaggag gtcatgcagt ggtttgggcc 721 gggcagctgc aaggaaaact ggttgccatc aaggccttcc caccgaggtc tgtggctcag 781 ttccaagctg agagagcatt gtacgaactt ccaggcctac agcacgacca cattgtccga 841 tttatcactg ccagccgggg gggtcctggc cgcctgctct ctgggcccct gctggtactg 901 gaactgcatc ccaagggctc cctgtgccac tacttgaccc agtacaccag tgactgggga 961 agttccctgc ggatggcact gtccctggcc cagggcctgg catttctcca tgaggagcgc1021 tggcagaatg gccaatataa accaggtatt gcccaccgag atctgagcag ccagaatgtg1081 ctcattcggg aagatggatc atgtgccatt ggagacctgg gccttgcctt ggtgctccct1141 ggcctcactc agccccctgc ctggacccct actcaaccac aaggcccagc tgccatcatg1201 gaagctggca cccagaggta catggcacca gagctcttgg acaagactct ggacctacag1261 gattggggca tggccctccg acgagctgat atttactctt tggctctgct cctgtgggag1321 atactgagcc gctgcccaga tttgaggcct gacagcagtc caccaccctt ccaactggcc1381 tatgaggcag aactgggcaa tacccctacc tctgatgagc tatgggcctt ggcagtgcag1441 gagaggaggc gtccctacat cccatccacc tggcgctgct ttgccacaga ccctgatggg1501 ctgagggagc tcctagaaga ctgttgggat gcagacccag aagcacggct gacagctgag1561 tgtgtacagc agcgcctggc tgccttggcc catcctcaag agagccaccc ctttccagag1621 agctgtccac gtggctgccc acctctctgc ccagaagact gtacttcaat tcctgcccct1681 accatcctcc cctgtaggcc tcagcggagt gcctgccact tcagcgttca gcaaggccct1741 tgttccagga atcctcagcc tgcctgtacc ctttctcctg tgtaaatatg cagtttatgt1801 gtcatcaatg tacatgccaa cataaatatg gcgattgtat agctgt

1-34. (canceled)
 35. The method of identifying an agent that modulatesMISRII-mediated signal transduction in a cell, comprising: a) providinga cell containing a nucleic acid construct comprising a nucleic acidsequence encoding a BMP-responsive element (BME) or fragment thereof,which is operatively linked to a nucleic acid sequence encoding areporter gene; and b) contacting the cell or an extract of the cell withan agent; and c) measuring the signal from the reporter gene, whereby achange in the signal from the reporter gene in the presence of the agentcompared to the signal from the reporter gene in the absence of theagent indicates the agent modulates MISRII-mediated signal transduction.36. The method of claim 35, wherein a change which is an increase in thesignal in step (c) indicates the agent activates or increases MISRIImediated signal transduction, or activates and increases MISRII mediatedsignal transduction.
 37. The method of claim 35, wherein a change whichis a decrease in the signal in step (c) indicates the agent inhibits ordecreases MISRII mediated signal transduction, or inhibits and decreasesMISRII mediated signal transduction.
 38. The method of claim 35, furthercomprising introducing a nucleic acid construct comprising a nucleicacid sequence encoding MISRII or a homologue or fragment thereof,wherein the nucleic acid sequence encoding MISRII is operatively linkedto a promoter.
 39. The method of claim 38, wherein the promoter is aconstitutive or inducible or tissue-specific promoter.
 40. The method ofclaim 35, wherein the BMP-responsive element is SEQ ID NO: 1 or afunctional fragment thereof.
 41. The method of claim 35, wherein thereporter gene encodes a protein with fluorescent and/or chromogenicactivity or variants or functional fragments thereof.
 42. The method ofclaim 41, wherein the chromogenic protein is the bioluminescent proteinor functional variants thereof.
 43. The method of claim 42, wherein thebioluminescent protein is luciferase or functional fragments or modifiedfunctional versions thereof.
 44. The method of claim 35, wherein theagent is a small molecule, nucleic acid, nucleic acid analogue, aptamer,ribosome, peptide, protein, antibody, or variants and functionalfragments thereof.
 45. The method of claim 44, wherein the antibody is arecombinant antibody, humanized antibody, chimeric antibody, modifiedantibody, monoclonal antibody, polyclonal antibody, miniantibody,dimeric miniantibody, minibody, diabody or tribody or functionalvariants, functional analogues or functional modified versions thereof.46. The method of claim 44, wherein the nucleic acid is DNA, RNA,nucleic acid analogue, peptide nucleic acid (PNA), pseudo-complementaryPNA (pcPNA), locked nucleic acid (LNA) or functional analogues thereof.47. The method of claim 46, wherein the RNA is a small inhibitory RNA,siRNA, microRNA, shRNA, miRNA and functional analogues and homologuesand functional variants thereof. 48.-79. (canceled)